PMC:2800179 / 13000-15386 JSON TXT

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GO-CC

{"project":"GO-CC","denotations":[{"id":"T6420","span":{"begin":28,"end":33},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T6421","span":{"begin":1045,"end":1050},"obj":"http://purl.obolibrary.org/obo/GO_0005623"},{"id":"T6422","span":{"begin":585,"end":593},"obj":"http://purl.obolibrary.org/obo/GO_0016020"},{"id":"T6423","span":{"begin":1410,"end":1418},"obj":"http://purl.obolibrary.org/obo/GO_0016020"},{"id":"T6424","span":{"begin":1458,"end":1467},"obj":"http://purl.obolibrary.org/obo/GO_0016020"},{"id":"T6425","span":{"begin":1554,"end":1563},"obj":"http://purl.obolibrary.org/obo/GO_0016020"},{"id":"T6426","span":{"begin":1791,"end":1800},"obj":"http://purl.obolibrary.org/obo/GO_0016020"},{"id":"T6427","span":{"begin":2324,"end":2333},"obj":"http://purl.obolibrary.org/obo/GO_0016020"},{"id":"T6428","span":{"begin":648,"end":656},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T6429","span":{"begin":749,"end":757},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T6430","span":{"begin":1612,"end":1620},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T6431","span":{"begin":1670,"end":1678},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T6432","span":{"begin":2093,"end":2101},"obj":"http://purl.obolibrary.org/obo/GO_0019815"},{"id":"T6433","span":{"begin":648,"end":656},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T6434","span":{"begin":749,"end":757},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T6435","span":{"begin":1612,"end":1620},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T6436","span":{"begin":1670,"end":1678},"obj":"http://purl.obolibrary.org/obo/GO_0042571"},{"id":"T6437","span":{"begin":2093,"end":2101},"obj":"http://purl.obolibrary.org/obo/GO_0042571"}],"text":"Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]."}

GO-MF

{"project":"GO-MF","denotations":[{"id":"T6413","span":{"begin":648,"end":656},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T6414","span":{"begin":749,"end":757},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T6415","span":{"begin":1612,"end":1620},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T6416","span":{"begin":1670,"end":1678},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T6417","span":{"begin":2093,"end":2101},"obj":"http://purl.obolibrary.org/obo/GO_0003823"},{"id":"T6418","span":{"begin":684,"end":686},"obj":"http://purl.obolibrary.org/obo/GO_0033968"},{"id":"T6419","span":{"begin":2364,"end":2367},"obj":"http://purl.obolibrary.org/obo/GO_0004707"}],"text":"Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]."}

GO-BP

{"project":"GO-BP","denotations":[{"id":"T6409","span":{"begin":174,"end":179},"obj":"http://purl.obolibrary.org/obo/GO_0019835"},{"id":"T6410","span":{"begin":334,"end":342},"obj":"http://purl.obolibrary.org/obo/GO_0007349"},{"id":"T6411","span":{"begin":684,"end":686},"obj":"http://purl.obolibrary.org/obo/GO_0033968"},{"id":"T6412","span":{"begin":2364,"end":2367},"obj":"http://purl.obolibrary.org/obo/GO_0004707"}],"text":"Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]."}

2_test

{"project":"2_test","denotations":[{"id":"20072622-19342628-93233369","span":{"begin":2382,"end":2384},"obj":"19342628"}],"text":"Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]."}

pmc-enju-pas

bionlp-st-ge-2016-spacy-parsed

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T5473","span":{"begin":1973,"end":1977},"obj":"http://purl.obolibrary.org/obo/UBERON_0001913"}],"text":"Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]."}

sentences

events-check-again

{"project":"events-check-again","denotations":[{"id":"T6440","span":{"begin":595,"end":600},"obj":"Protein"}],"text":"Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]."}

bionlp-st-ge-2016-reference-tees

{"project":"bionlp-st-ge-2016-reference-tees","denotations":[{"id":"T6441","span":{"begin":447,"end":450},"obj":"Protein"},{"id":"T6442","span":{"begin":595,"end":608},"obj":"Protein"},{"id":"T6443","span":{"begin":702,"end":724},"obj":"Protein"},{"id":"T6444","span":{"begin":808,"end":811},"obj":"Protein"},{"id":"T6445","span":{"begin":969,"end":978},"obj":"Protein"},{"id":"T6446","span":{"begin":1024,"end":1037},"obj":"Protein"},{"id":"T6447","span":{"begin":1652,"end":1683},"obj":"Protein"},{"id":"T6448","span":{"begin":1684,"end":1689},"obj":"Protein"},{"id":"T6449","span":{"begin":1690,"end":1701},"obj":"Protein"},{"id":"T6450","span":{"begin":1716,"end":1738},"obj":"Protein"},{"id":"T6451","span":{"begin":1912,"end":1955},"obj":"Protein"},{"id":"T6452","span":{"begin":2364,"end":2367},"obj":"Protein"}],"text":"Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]."}

bionlp-st-ge-2016-reference

{"project":"bionlp-st-ge-2016-reference","denotations":[{"id":"T5474","span":{"begin":595,"end":600},"obj":"Protein"}],"namespaces":[{"prefix":"_base","uri":"http://bionlp.dbcls.jp/ontology/ge.owl#"}],"text":"Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]."}

bionlp-st-ge-2016-uniprot

{"project":"bionlp-st-ge-2016-uniprot","denotations":[{"id":"T5928","span":{"begin":201,"end":203},"obj":"P0A7Z4"},{"id":"T5929","span":{"begin":1690,"end":1693},"obj":"P19838"},{"id":"T5930","span":{"begin":1690,"end":1701},"obj":"P19838"},{"id":"T5931","span":{"begin":1727,"end":1730},"obj":"Q04206"},{"id":"T5932","span":{"begin":1727,"end":1730},"obj":"P21579"}],"namespaces":[{"prefix":"_base","uri":"http://www.uniprot.org/uniprot/"}],"text":"Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]."}

test2

{"project":"test2","denotations":[{"id":"T5472","span":{"begin":595,"end":600},"obj":"Protein"}],"text":"Western Blot Analysis\nTHP-1 cells were stimulated for various lengths of time with 0.1 mg/ml C10 or C40, or 10 µg/ml LPS. Cells were then pelleted, washed and homogenised in lysis buffer (10 mM Hepes, pH 7.9, 150 mM NaCl, 1 mM EDTA, 0.6% NP-40, and 0.5 mM PMSF) on ice. Homogenates were sonicated, centrifuged at 10,000 rpm to remove cellular debris, and supernatant collected. Protein concentration was determined using the DC Protein Assay (Bio-Rad). Proteins in samples (15 µg total proteins) were resolved in a denaturing 12% polyacrylamide gel and transferred to a nitrocellulose membrane. I-κBα protein was detected using a rabbit polyclonal antibody (Santa Cruz Biotechnology, CA) followed by a horseradish peroxidase-coupled goat polyclonal antibody against rabbit Ig (Caltag Laboratories). Finally, IκB bands were revealed using the ECL™ detection system (Amersham Pharmacia Biotech, Les Ullis, France) according to the manufacturers' instruction. Antibody to α-Tubulin (Santa Cruz) was use as loading control.\nFor nuclear NF-κB, THP-1 cells were stimulated with 1 mg/ml C10 or C40 for 30 minutes at 37°C. Cells were then pelleted and nuclei separated as described [31]. Nuclei were washed and homogenized directly in loading (Laemli) buffer and heated for 5 minutes at 100°C. Proteins in samples were resolved in a denaturing 8% polyacrylamide gel and transferred to a polyvinylidine fluoride (PVDF) membrane (Immobilon-P; Millipore, Bedford, MA). Membranes were incubated in blocking buffer (1% BSA, in PBS) for two hours at room temperature. Membranes were subsequently probed with the corresponding antibody in blocking buffer, overnight. Rabbit polyclonal antibody anti-NF-κB p50 subunit (# sc-114) or anti-NF-κB p65 subunit (# sc-109) from Santa Cruz Biotechnology were used. Membranes were washed six times in PBS with 0.05% Tween 20, 5 minutes each time, and incubated with a 1/3000 dilution of HRP-conjugated F(ab')2 goat anti-rabbit IgG in 5% nonfat dry milk and 0.05% Tween 20 in PBS for 1 hour at room temperature. After washing six more times in PBS with 0.05% Tween 20, antibody-reactive proteins were detected using a chemiluminescence substrate (SuperSignal; Pierce, Rockford, IL) according to the manufacturer's instructions. To confirm that equivalent amounts of protein were loaded in each line, membranes were also Western blotted for ERK as described [32]."}

PMC:2800179 / 13000-15386 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:2800179 / 13000-15386 JSON TXT