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2_test

{"project":"2_test","denotations":[{"id":"22529521-8455207-67992085","span":{"begin":1577,"end":1578},"obj":"8455207"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

pmc-enju-pas

bionlp-st-ge-2016-test-proteins

{"project":"bionlp-st-ge-2016-test-proteins","denotations":[{"id":"T3109","span":{"begin":4964,"end":4971},"obj":"Protein"},{"id":"T4389","span":{"begin":8514,"end":8518},"obj":"Protein"},{"id":"T4388","span":{"begin":8258,"end":8262},"obj":"Protein"},{"id":"T4071","span":{"begin":6837,"end":6841},"obj":"Protein"},{"id":"T4070","span":{"begin":6822,"end":6835},"obj":"Protein"},{"id":"T4069","span":{"begin":6810,"end":6815},"obj":"Protein"},{"id":"T4068","span":{"begin":6790,"end":6808},"obj":"Protein"},{"id":"T3385","span":{"begin":5890,"end":5894},"obj":"Protein"}],"namespaces":[{"prefix":"_base","uri":"http://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

bionlp-st-ge-2016-uniprot

{"project":"bionlp-st-ge-2016-uniprot","denotations":[{"id":"T4291","span":{"begin":6834,"end":6839},"obj":"http://www.uniprot.org/uniprot/P05231"},{"id":"T4290","span":{"begin":6810,"end":6815},"obj":"http://www.uniprot.org/uniprot/P01584"},{"id":"T4289","span":{"begin":6782,"end":6785},"obj":"http://www.uniprot.org/uniprot/P01375"},{"id":"T3688","span":{"begin":6475,"end":6482},"obj":"http://www.uniprot.org/uniprot/P60709"},{"id":"T3687","span":{"begin":5791,"end":5798},"obj":"http://www.uniprot.org/uniprot/P60709"},{"id":"T3226","span":{"begin":4964,"end":4971},"obj":"http://www.uniprot.org/uniprot/P60709"}],"namespaces":[{"prefix":"_base","uri":"http://www.uniprot.org/uniprot/"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T3373","span":{"begin":5706,"end":5710},"obj":"http://purl.obolibrary.org/obo/UBERON_0001913"},{"id":"T2308","span":{"begin":3440,"end":3445},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T1223","span":{"begin":605,"end":611},"obj":"http://purl.obolibrary.org/obo/UBERON_0000479"},{"id":"T1222","span":{"begin":453,"end":459},"obj":"http://purl.obolibrary.org/obo/UBERON_0000479"},{"id":"T1221","span":{"begin":427,"end":434},"obj":"http://purl.obolibrary.org/obo/UBERON_0000055"},{"id":"T1220","span":{"begin":421,"end":434},"obj":"http://purl.obolibrary.org/obo/UBERON_0001981"},{"id":"T1219","span":{"begin":421,"end":426},"obj":"http://purl.obolibrary.org/obo/UBERON_0000178"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

GO-BP

GO-MF

{"project":"GO-MF","denotations":[{"id":"T4284","span":{"begin":7560,"end":7562},"obj":"http://purl.obolibrary.org/obo/GO_0033968"},{"id":"T4283","span":{"begin":6972,"end":6974},"obj":"http://purl.obolibrary.org/obo/GO_0033968"},{"id":"T4282","span":{"begin":6834,"end":6839},"obj":"http://purl.obolibrary.org/obo/GO_0005138"},{"id":"T3674","span":{"begin":6191,"end":6199},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T3673","span":{"begin":5963,"end":5971},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T3672","span":{"begin":5764,"end":5774},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T2665","span":{"begin":4348,"end":4359},"obj":"http://purl.obolibrary.org/obo/GO_0003677"},{"id":"T2664","span":{"begin":4352,"end":4359},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T2663","span":{"begin":3797,"end":3804},"obj":"http://purl.obolibrary.org/obo/GO_0005488"},{"id":"T3676","span":{"begin":5932,"end":5934},"obj":"http://purl.obolibrary.org/obo/GO_0033968"},{"id":"T3675","span":{"begin":5837,"end":5839},"obj":"http://purl.obolibrary.org/obo/GO_0033968"},{"id":"T1500","span":{"begin":1374,"end":1376},"obj":"http://purl.obolibrary.org/obo/GO_0033968"},{"id":"T3224","span":{"begin":4466,"end":4468},"obj":"http://purl.obolibrary.org/obo/GO_0033968"},{"id":"T3223","span":{"begin":4555,"end":4557},"obj":"http://purl.obolibrary.org/obo/GO_0003964"},{"id":"T3222","span":{"begin":4411,"end":4413},"obj":"http://purl.obolibrary.org/obo/GO_0003964"},{"id":"T2163","span":{"begin":2484,"end":2486},"obj":"http://purl.obolibrary.org/obo/GO_0033968"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

GO-CC

sentences

ICD10

{"project":"ICD10","denotations":[{"id":"T1918","span":{"begin":1531,"end":1537},"obj":"http://purl.bioontology.org/ontology/ICD10/T14.9"},{"id":"T1917","span":{"begin":1506,"end":1512},"obj":"http://purl.bioontology.org/ontology/ICD10/T14.9"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

simple1

{"project":"simple1","denotations":[{"id":"T4551","span":{"begin":8514,"end":8518},"obj":"Protein"},{"id":"T4550","span":{"begin":8258,"end":8262},"obj":"Protein"},{"id":"T3225","span":{"begin":4964,"end":4971},"obj":"Protein"},{"id":"T4288","span":{"begin":6837,"end":6841},"obj":"Protein"},{"id":"T4287","span":{"begin":6822,"end":6835},"obj":"Protein"},{"id":"T4286","span":{"begin":6810,"end":6815},"obj":"Protein"},{"id":"T4285","span":{"begin":6790,"end":6808},"obj":"Protein"},{"id":"T3686","span":{"begin":5890,"end":5894},"obj":"Protein"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

BioNLP16_DUT

{"project":"BioNLP16_DUT","denotations":[{"id":"T4732","span":{"begin":8514,"end":8518},"obj":"Protein"},{"id":"T4731","span":{"begin":8258,"end":8262},"obj":"Protein"},{"id":"T4337","span":{"begin":6837,"end":6841},"obj":"Protein"},{"id":"T4336","span":{"begin":6822,"end":6835},"obj":"Protein"},{"id":"T4335","span":{"begin":6810,"end":6815},"obj":"Protein"},{"id":"T4334","span":{"begin":6790,"end":6808},"obj":"Protein"},{"id":"T3997","span":{"begin":5890,"end":5894},"obj":"Protein"},{"id":"T3353","span":{"begin":4964,"end":4971},"obj":"Protein"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

BioNLP16_Messiy

{"project":"BioNLP16_Messiy","denotations":[{"id":"T4299","span":{"begin":6837,"end":6841},"obj":"Protein"},{"id":"T4298","span":{"begin":6822,"end":6835},"obj":"Protein"},{"id":"T4297","span":{"begin":6810,"end":6815},"obj":"Protein"},{"id":"T4296","span":{"begin":6790,"end":6808},"obj":"Protein"},{"id":"T4556","span":{"begin":8266,"end":8272},"obj":"Protein_catabolism"},{"id":"T4555","span":{"begin":8514,"end":8518},"obj":"Protein"},{"id":"T4554","span":{"begin":8258,"end":8262},"obj":"Protein"},{"id":"T3690","span":{"begin":5890,"end":5894},"obj":"Protein"},{"id":"T3228","span":{"begin":4964,"end":4971},"obj":"Protein"}],"relations":[{"id":"R3872","pred":"themeOf","subj":"T4554","obj":"T4556"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

DLUT931

{"project":"DLUT931","denotations":[{"id":"T4295","span":{"begin":6837,"end":6841},"obj":"Protein"},{"id":"T4294","span":{"begin":6822,"end":6835},"obj":"Protein"},{"id":"T4293","span":{"begin":6810,"end":6815},"obj":"Protein"},{"id":"T4292","span":{"begin":6790,"end":6808},"obj":"Protein"},{"id":"T4553","span":{"begin":8514,"end":8518},"obj":"Protein"},{"id":"T4552","span":{"begin":8258,"end":8262},"obj":"Protein"},{"id":"T3689","span":{"begin":5890,"end":5894},"obj":"Protein"},{"id":"T3227","span":{"begin":4964,"end":4971},"obj":"Protein"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

bionlp-st-ge-2016-test-ihmc

bionlp-st-ge-2016-spacy-parsed

bionlp-st-ge-2016-test-tees

testone

{"project":"testone","denotations":[{"id":"T4373","span":{"begin":8514,"end":8518},"obj":"Protein"},{"id":"T4372","span":{"begin":8258,"end":8262},"obj":"Protein"},{"id":"T4043","span":{"begin":6837,"end":6841},"obj":"Protein"},{"id":"T4042","span":{"begin":6822,"end":6835},"obj":"Protein"},{"id":"T4041","span":{"begin":6810,"end":6815},"obj":"Protein"},{"id":"T4040","span":{"begin":6790,"end":6808},"obj":"Protein"},{"id":"T3097","span":{"begin":4964,"end":4971},"obj":"Protein"},{"id":"T3370","span":{"begin":5890,"end":5894},"obj":"Protein"},{"id":"T2032","span":{"begin":2556,"end":2565},"obj":"Gene_expression"}],"relations":[{"id":"R3486","pred":"equivalentTo","subj":"T4041","obj":"T4040"},{"id":"R3487","pred":"equivalentTo","subj":"T4043","obj":"T4042"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

test3

{"project":"test3","denotations":[{"id":"T4377","span":{"begin":8514,"end":8518},"obj":"Protein"},{"id":"T4376","span":{"begin":8258,"end":8262},"obj":"Protein"},{"id":"T4375","span":{"begin":8514,"end":8518},"obj":"Protein"},{"id":"T4374","span":{"begin":8258,"end":8262},"obj":"Protein"},{"id":"T4051","span":{"begin":6837,"end":6841},"obj":"Protein"},{"id":"T4050","span":{"begin":6822,"end":6835},"obj":"Protein"},{"id":"T4049","span":{"begin":6810,"end":6815},"obj":"Protein"},{"id":"T4048","span":{"begin":6790,"end":6808},"obj":"Protein"},{"id":"T4047","span":{"begin":6837,"end":6841},"obj":"Protein"},{"id":"T4046","span":{"begin":6822,"end":6835},"obj":"Protein"},{"id":"T4045","span":{"begin":6810,"end":6815},"obj":"Protein"},{"id":"T4044","span":{"begin":6790,"end":6808},"obj":"Protein"},{"id":"T3099","span":{"begin":4964,"end":4971},"obj":"Protein"},{"id":"T3098","span":{"begin":4964,"end":4971},"obj":"Protein"},{"id":"T3372","span":{"begin":5890,"end":5894},"obj":"Protein"},{"id":"T3371","span":{"begin":5890,"end":5894},"obj":"Protein"}],"relations":[{"id":"R3488","pred":"equivalentTo","subj":"T4049","obj":"T4048"},{"id":"R3489","pred":"equivalentTo","subj":"T4051","obj":"T4050"}],"text":"2. Methods and Materials\n\n2.1. Primary Culture and Identification of Mouse Astrocytes\nNrf2 knockout ICR mice were kindly provided by Dr. Thomas W. Kensler (Johns Hopkins University, Baltimore, MD, USA). Primary astrocytes were obtained from postnatal 2-day-old Nrf2 wide type (WT) and knockout (KO) mice (6 mice for each genotype). Following decapitation, the cortices were dissected out, and the meninges and associated blood vessels were removed. The tissue was roughly chopped with a scalpel blade, incubated in 0.5% trypsin for 10 minutes at 37°C, and agitated every few minutes. After digestion, the tissue was rinsed twice in DMEM with 10% FBS, followed by a mechanical dissociation in DMEM with 20% FBS and 5 units/mL penicillin, 5 μg/mL streptomycin (complete culture medium). After incubation for 1 h, the supernatant was transferred to a new flask or dish (Costar, USA) for depleting the residual epithelial cells. Then the cells were cultivated at 37°C with 5% CO2. And the complete culture medium was half-changed twice a week. Astrocytes expanded for about 7 days to reach confluence. Then the flasks or dishes were shaken at 150 rpm for 4 h to deplete the microglia and less adherent cells from the cultures. After shaking, the resulting cultures were mainly astrocytes, which were determined by immunoreactivity for GFAP (sc-166481, Santa Cruz Biotechnology, CA, USA). Cells passaged for 2-3 generations were used in the following studies.\n\n2.2. In Vitro Model of TBI Established by Scratch Injury\nAstrocyte scratch injury was performed as in a previous report [7]. Astrocytes were planted in 6-well plates and grown to confluence. The cell monolayer was scratched with a sterile 26G syringe needle, resulting in the formation of a 0.5 mm wide gap. Immediately after scratch, cells were washed twice with sterile PBS, cultured with complete culture medium, and named as TBI group. Cells, which did not received scratch, were used as blank control and named as sham group.\n\n2.3. Cell Death Measurement by CCK-8 Assay\n1 × 103/well cells were seeded to 96-well culture plates and cultivated for 24 h to adhere. Then the cell was scrated as mentioned above. Astrocyte cell death was assessed by cell counting kit-8 (CCK-8) (Dojindo, Japan) assay 24 h after scratch according to the manufacturer's protocol. Briefly, 10 μL CCK-8 was added into every well and incubated for 1 h. Then OD value was read at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). All measurements were performed in sextuplicate. Results were expressed as mean of OD value at 450 nm ± SD.\n\n2.4. Electrophoretic Mobility Shift Assay (EMSA)\nMonolayers of astrocytes were washed with PBS and harvested by scraping into cold PBS. The cell pellet obtained by centrifugation was resuspended in buffer containing 10 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1.0 mM DTT, and 0.5 mM phenylmethylsulfonyl fluoride. Then 10% Nonidet P-40 was added and vortexed briefly, and the nuclei were pelleted by centrifugation. The nuclear proteins were extracted with buffer containing 20 mM HEPES (pH 7.9), 0.4 mM NaCl, 1.0 mM EDTA, 1.0 mM EGTA, 1.0 mM DTT, and 1.0 mM phenylmethylsulfonyl fluoride. Insoluble material was removed by centrifugation at 14000 rpm, and the supernatant containing the nuclear proteins was stored at –80°C until use. Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). Levels of NF-κB DNA binding activity were quantified by software ImageJ.\n\n2.5. RT-PCR\nTotal RNA was isolated with Trizol (Invitrogen, CA, USA), and single-stranded cDNA was synthesized from 2 μg of total RNA with BU-Script RT-Kit (Biunique, Jiangsu, China) according to the manufacturer's protocol. The cDNA was stored in −20°C. Reverse transcription was conducted with GoTaq Green Master Mix (Promega, WI, USA) according to the manufacturer's protocol. Table 1 shows the primers and PCR parameters. PCR products were detected by agarose gel electrophoresis. The intensity of the bands was analyzed by ImageJ program. The level of β-actin was used as an internal standard.\n\n2.6. Western Blot\nTo obtain total protein lysates, cells were homogenized in RIPA buffer (1% NP40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM EGTA, 1 mM Na3VO4, 20 mM NaF, 0.5 mM DTT, 1 mM PMSF, and protease inhibitor cocktail in PBS pH 7.4) and centrifuged at 12,000 g for 15 min at 4°C. Protein concentrations were estimated by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Fifty micrograms of the resulting cytosolic protein extracts were heat-denatured in Laemmli sample loading buffer, separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and electroblotted onto a nitrocellulose membrane. For immunoblotting, membranes were blocked with 5% nonfat dry milk in saline buffer overnight at 4°C, and the following antibodies were used: anti-β-actin (sc-130657, Santa Cruz Biotechnology, CA, USA, 43 kDa) and anti-matrix metallopeptidase 9 (MMP9) (sc-6841, Santa Cruz Biotechnology, CA, USA, 92 kDa). Each primary antibody was diluted appropriately in blocking buffer and then added to the blots for 1 h at room temperature. The blots were washed three times in the washing buffer and covered with the horseradish peroxidase-linked secondary antibody at a 1 : 2000 dilution for 1 h. Blots were incubated with enhanced chemiluminescence (ECL) detection system (Amersham Biosciences, Bucks, UK) and exposed to radiographic film (Fuji Hyperfilm, Tokyo, Japan). ImageJ was used to analyze the intensity of the blots. The level of β-actin was used as internal standard.\n\n2.7. Enzyme-Linked Immunosorbent Assay (ELISA)\nCells of four groups were homogenized as mentioned above. The supernatant was collected, and total protein was determined by Coomassie Plus Protein Assay Reagent (Pierce, IL, USA). Levels of tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and interleukin-6 (IL-6) protein were quantified using ELISA kits specific for mouse according to the manufacturer's instructions (Bender MedSystems Inc. CA, USA). Briefly, prepared the standard and created the standard dilution for building standard curve. Then samples and biotinconjugate were added to microwell strips. After incubated for 2 h at room temperature, the microwell strips were washed 3 times with wash buffer, and streptavidin-HRP were added to all wells. After incubated for 1 h, microwell strips were washed 3 times followed by adding TMB substrate. After incubated for about 10–30 min, the stop solution was added. The colour intensity was measured at 450 nm using a Bio-Rad ELISA microplate reader (Bio-Rad Laboratories, CA, USA). The concentration of protein was determined according to the standard curve and expressed as pg/mg of total protein.\n\n2.8. Gelatine Zymography\nCells of four groups were homogenized in lysis buffer containing 50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM CaCl2, 0.2 mM NaN3, and 0.01% Triton. Soluble extracts were separated by centrifugation and stored at –20°C. Gelatin zymography was performed according to the manufacturer's instructions (Genmed Scientifics Inc, MA, USA). Briefly, 40 μg cytosolic protein extracts were separated by electrophoresis. Then the proteins were renatured by incubation in 2.5% Triton X-100 and then incubated in substrate buffer for 40 h at 37°C to enable the MMP9 to cleave the gelatin. After rinsing in water, each gel was stained with Coomassie blue for 1 h and destained in 50% methanol. Proteolytic activities were showed by clear bands in blue gel which indicates the lysis of the substrate. Quantification of MMP9 band density was performed with image analysis program ImageJ.\n\n2.9. Statistical Analysis\nData were expressed as mean ± SD and evaluated by ANOVA and LSD multiple comparison test. P values \u003c0.05 were considered to be significant. All analyses were performed by using SPSS 18.0 software."}

PMC:3317373 / 2853-11658 JSONTXT

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PMC:3317373 / 2853-11658 JSON TXT