PMC:2222968 / 35148-46702 JSON TXT

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2_test

{"project":"2_test","denotations":[{"id":"18162042-11441076-84705058","span":{"begin":315,"end":317},"obj":"11441076"},{"id":"18162042-12554698-84705059","span":{"begin":812,"end":814},"obj":"12554698"},{"id":"18162042-16387593-84705060","span":{"begin":1405,"end":1407},"obj":"16387593"},{"id":"18162042-10037236-84705061","span":{"begin":2883,"end":2885},"obj":"10037236"},{"id":"18162042-16517728-84705062","span":{"begin":3895,"end":3897},"obj":"16517728"},{"id":"18162042-17298430-84705063","span":{"begin":5347,"end":5349},"obj":"17298430"},{"id":"T68613","span":{"begin":315,"end":317},"obj":"11441076"},{"id":"T44281","span":{"begin":812,"end":814},"obj":"12554698"},{"id":"T86850","span":{"begin":1405,"end":1407},"obj":"16387593"},{"id":"T11976","span":{"begin":2883,"end":2885},"obj":"10037236"},{"id":"T56825","span":{"begin":3895,"end":3897},"obj":"16517728"},{"id":"T99961","span":{"begin":5347,"end":5349},"obj":"17298430"}],"text":"Materials and Methods\n\nMice.\nNormal B6 mice were purchased from the Jackson Laboratories (Bar Harbor, Maine). Transgenic DO11.10 mice, expressing a T cell receptor for OVA323–339 peptide in the context of H-2d, were backcrossed with mice expressing GATA-3, driven by the human CD2 locus control region (CD2-GATA3) [22], resulting in DO11.10xCD2-GATA3 mice. Mice used for experiments were backcrossed on a BALB/c background for a minimum of eight generations and used at an age of 8–12 wk. Mice were housed under specific pathogen-free conditions and all animal studies were performed according to institutional and state guidelines.\n\nIsolation of CD4+ T cells.\nCD4+ T cells were isolated from blood of healthy human volunteers using the anti-CD4 magnetic beads (Dynal, Hamburg, Germany) as previously described [60]. The purity of CD4+ T cells was initially tested by FACS and was ≥ 95%. Monoclonality of T cell clones was confirmed by TCR-chain mapping and was identified to be Vbeta8 positive. The clones were characterized by high IL-4 secretion.\n\nQuantitative real-time PCR.\nThe PCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer 5′-GAA ACA GCA CAT TCC CAG AGT TC-3′; FOXP3 reverse primer 5′-ATG GCC CAG CGG ATG AG-3′; EF-1α forward primer and reverse primer as described [61]; GATA3 forward primer 5′-GCG GGC TCT ATC ACA AAA TGA-3′ and rwd 5′-GCT CTC CTG GCT GCA GAC AGC-3′. The prepared cDNAs were amplified using SYBR-PCR mastermix (Biorad) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).\nQuantitative PCR of murine samples was performed with Brilliant SYBR Green QPCR master mix (Stratagene) and the following primers: Ubiquitin C, 5′- AGG TCA AAC AGG AAG ACA GAC GTA-3′ and 5′-TCACACCCAAGAACAAGCACA-3′; Smad-7, 5′-GAA ACC GGG GGA ACG AAT TAT-3′ and 5′-CGC GAG TCT TCT CCT CCC A-3′; TGF-ß1, 5′-TGA CGT CAC TGG AGT TGT ACG G-3′ and 5′-GGT TCA TGT CAT GGA TGG TGC-3′. Primer pairs were evaluated for integrity by analysis of the amplification plot, dissociation curves, and efficiency of PCR amplification. PCR conditions were 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 °C for 1 min using an 7300 real-time PCR system (Applied Biosystems). PCR amplification of the housekeeping gene encoding ubiquitin C was performed during each run for each sample to allow normalization between samples. Relative quantification and calculation of the range of confidence was performed using the comparative ΔΔCT method.\n\nInducible murine Treg culture.\nNaive CD4+ T cells (CD4+, CD62L+, and CD25–) were isolated from pooled lymph nodes and spleens by FACS (FACS Aria, BD Biosciences). 5 × 105 T cells were co-cultured with 2.5 × 104 bone marrow–derived dendritic cells [62] and 0.01 μg/ml OVA323–339 peptide (Ansynth) in the presence or absence of 20-ng/μl rhTGF-ß1 (Peprotech) in 48-well plates. After 4 d, cells were harvested and analyzed for intracellular FOXP3 expression by FACS or gene expression by quantitative RT-PCR.\n\nIn vitro T cell differentiation.\nCD4+ CD45RA+ magnetically-sorted (CD45RO depletion, MACS, according to the protocol of the manufacturer) cells were stimulated with immobilized plate-bound anti-CD3 (1 μg/ml, Okt3, IgG1) and anti-CD28 (2 μg/ml) in Th1 conditions: 25 ng/ml IL-12, 5 μg/ml anti-IL-4 (R\u0026D systems); in Th2 conditions: 25 ng/ml IL-4, 5 μg/ml anti-IFN-γ, 5 μg/ml anti-IL-12 (R\u0026D systems); or in Treg conditions: 10 ng/ml TGF-β, 5 μg/ml anti-IFN-γ, 5 μg/ml anti-IL-12, 5 μg/ml anti-IL-4. Proliferating cells were expanded in medium containing IL-2 (30 ng/ml).\n\nCloning of the FOXP3 promoter and construction of mutant constructs.\nThe FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate the pGL3 FOXP3 −511/+176 [24]. Site-directed mutagenesis in the FOXP3 promoter region were introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions. The following primer and its complementary strand were used: GTT TCT CAT GAG CCC TAT TAA GTC ATT CTT ACC TCT CAC CTC TGT GGT GA.\n\nTransfections and reporter gene assays.\nT cells were rested in serum-free AIM-V medium (Life Technologies) overnight. 3.5 μg of the FOXP3 promoter luciferase reporter vector and 0.5 μg phRL-TK were added to 3 × 106 CD4+ T cells resuspended in 100 μL of Nucleofector solution (Amaxa Biosystems) and electroporated using the U-15 program of the Nucleofector. After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega Biotech) according to the manufacturer's instructions. Data were normalized by the activity of renilla luciferase.\n\nRNA isolation and cDNA synthesis.\nRNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Reverse transcription of human samples was performed with TaqMan reverse-transcription reagents (Applied Biosystems) with random hexamers according to the manufacturer's protocol.\n\nRecombinant TAT proteins.\nThe cDNAs encoding GATA3 protein or the truncated GATA3 (lacking the two zinc fingers) were cloned in frame into an expression vector along with the TAT sequence as previously described [63]. Proteins were expressed in BL21 Star (DE3)pLysS (Invitrogen) and lysates were purified by Ni2+-chelate column chromatography. Both TAT-linked proteins were more than 95% pure, based on Coomassie blue staining of sodium disulfate acrylamide gels.\n\nTAT-GATA3 transduction.\nCD4+CD45RA+ cells were cultured in AIMV medium and transduced with 20 nM, 10 nM, or 500 nM of full-length or truncated GATA3 over the course of 4 h. After 4 h, the cells were washed and activated with soluble anti-CD3 and anti-CD28 and TGF-β (10 ng/ml). Each day, the TAT proteins were freshly added to the medium. FOXP3 expression was measured after 5 d by intracellular staining.\n\nIntracellular cytokine staining.\nT cells were stimulated with 2 × 10−7 M PMA and 1 μg/ml of ionomycin (Sigma Chemicals) for 4 h. The following mAb was used: anti-IL-4-PE (8D4–8, BD). Matched isotype controls were used at the same protein concentration as the respective antibodies. Four-color FACS was performed using an EPICS XL-MCL (Beckman Coulter) using the software Expo32 version for data acquisition and evaluation.\n\nFlow cytometry.\nFor analysis of FOXP3 expression at the single-cell level, cells were first stained with the monoclonal antibody CD25 (Beckman Coulter), and after fixation and permeabilization, cells were incubated with PE-conjugated monoclonal antibody PCH101 (anti-human FOXP3; eBioscience) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL). For cell surface marker staining, cells were incubated for 20 min at 4°C in staining buffer with the following antibodies: anti–CD152-PE (CTLA-4; BD), anti–PD-1 (eBiosciences), anti-GITR (R \u0026 D Systems), anti-CD69 (Beckman Coulter), anti-CD103 (DakoCytomation), anti-CD62L (Beckman Coulter), or anti-HLA-DR (Beckman Coulter). The controls were FITC, PE, or ECD-conjugated mouse IgG1 or rat IgG2a. For staining of mouse cells, the following mAbs from BD Biosciences were used following standard techniques as described above: anti-CD3, anti-CD4, and anti-CD25. Anti-FcγRII/III antibody (2.4G2, ATCC) was included in all stainings to reduce nonspecific antibody binding. To isolate naive murine CD4 T cells from murine DO11.10 or DO11.10xCD2-GATA3 T cells, cells were stained with anti-CD25-FITC, anti-CD62L-PE, and anti-CD4-APC prior to sorting. Dead cells were excluded with 4′',6-Diamidino-2-phenylindole (DAPI). To analyze murine Foxp3 expression in inducible Treg cultures, cells were stained intracellularly with anti-Foxp3-PE according to manufacturer's instruction, in conjunction with anti-CD4-APC and LIVE/DEAD fixable dead cell stain kit (Invitrogen) to discriminate live cells. All monoclonal antibodies for murine cell stainings were purchased from eBioscience or BD Biosciences.\n\nWestern blotting.\nFor FOXP3 analysis on the protein level, 1 × 106 CD4+CD25– cells were lysed and loaded next to a protein-mass ladder (Magicmark, Invitrogen) on a NuPAGE 4–12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Life Science). Unspecific binding was blocked with BSA, and the membranes were subsequently incubated with an 1:200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4 °C. The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji). Membranes were incubated in stripping buffer and re-blocked for 1 h. The membranes were re-probed using anti-GATA3 (HG3–31; Santa Cruz Biotechnology), anti-T-bet (4B10, Santa Cruz Biotechnology), anti-GAPDH (6C5, Ambion), anti-phospho-SMAD2 (138D4), anti-phospho-STAT6 (5A4), and anti-STAT6 (Cell Signaling Technology),\n\nAdministration of cytokines and antibodies in vivo.\nAge- and gender-matched normal B6 mice received every other day intraperitoneal (ip) injections of PBS, 1.5 μg rmIL-4, 50 μg anti-IL-4 mAb (11B11 or MAB404), or a mixture of 1.5 μg rmIL-4 plus 50 μg anti-IL-4 mAb (11B11 or MAB404) for 7 d. Thereafter, spleen and lymph node cells were analyzed by flow cytometry for CD3, CD4, and CD25 expression. The anti-mouse IL-4 mAb MAB404 was obtained from R\u0026D Systems, the second anti-mouse IL-4 mAb 11B11 was purchased from eBioscience.\n\nChIP.\nChIP analysis was performed according to the manufacturer's protocol (Upstate Biotechnology) with the following modifications. iTreg and Th2 cells were fixed with 1% formaldehyde for 10 min at room temperature. The chromatin was sheared to 200-1000 bp of length by sonication with five pulses of 10 s at 30% power (Bandelin). The chromatin was pre-cleared for 2 h with normal mouse IgG beads and then incubated with anti-GATA3-agarose beads (HG3–31; Santa Cruz Biotechnology) for 2 h. Washing and elution buffers were used according to the protocol of Upstate Biotechnology. Crosslinks were reversed by incubation at 65 °C for 4 h in the presence of 0.2 M NaCl, and the DNA was purified by phenol/chloroform extraction. The amount of DNA was determined by conventional PCR. The PCR addressed for the FOXP3 promoter region −246 to −511 and was performed using the following primers: 5′-gtgccctttacgagt catctg-3′ and 5′-gtgccctttacgagtcatctg-3′. The PCR products were visualized using an ethidium bromide gel.\n\nPull-down assay.\nCD4+ T cells were stimulated with PMA and ionomycin for 2 h at 37°C. The cells were pelleted, resuspended in buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, protease inhibitors [Sigma]. and 0.1% NP-40) and lysed on ice for 15 min. Insoluble material was removed by centrifugation. The supernatant was diluted 1:3 with buffer D (as buffer C, but without NaCl). The lysates were incubated with 10 μg of poly(dI-dC) (Sigma) and 70 μl of streptavidin-agarose (Amersham Biosciences) carrying biotinylated oligonucleotides, for 3 h at 4 °C. The beads were washed twice with buffer C:D (1:3) and resuspended in DTT-containing loading buffer (NuPAGE; Invitrogen), heated to 70 °C for 10 min, and the eluants on a NuPAGE 4–12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Biosciences) and detected using an anti-GATA3 mAb (Santa Cruz Biotechnology). Accumulated signals were analyzed using AIDA software (Raytest).\n"}

pmc-enju-pas

bionlp-st-ge-2016-spacy-parsed

UBERON-AE

{"project":"UBERON-AE","denotations":[{"id":"T18065","span":{"begin":693,"end":698},"obj":"http://purl.obolibrary.org/obo/UBERON_0000178"},{"id":"T18776","span":{"begin":2737,"end":2742},"obj":"http://purl.obolibrary.org/obo/UBERON_0002391"},{"id":"T18777","span":{"begin":2737,"end":2748},"obj":"http://purl.obolibrary.org/obo/UBERON_0000029"},{"id":"T18778","span":{"begin":2753,"end":2760},"obj":"http://purl.obolibrary.org/obo/UBERON_0002106"},{"id":"T18779","span":{"begin":2846,"end":2857},"obj":"http://purl.obolibrary.org/obo/UBERON_0002371"},{"id":"T19753","span":{"begin":4250,"end":4255},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T19754","span":{"begin":4568,"end":4573},"obj":"http://purl.obolibrary.org/obo/UBERON_0001977"},{"id":"T22241","span":{"begin":9309,"end":9315},"obj":"http://purl.obolibrary.org/obo/UBERON_0002106"},{"id":"T22242","span":{"begin":9320,"end":9325},"obj":"http://purl.obolibrary.org/obo/UBERON_0002391"},{"id":"T22243","span":{"begin":9320,"end":9330},"obj":"http://purl.obolibrary.org/obo/UBERON_0000029"}],"text":"Materials and Methods\n\nMice.\nNormal B6 mice were purchased from the Jackson Laboratories (Bar Harbor, Maine). Transgenic DO11.10 mice, expressing a T cell receptor for OVA323–339 peptide in the context of H-2d, were backcrossed with mice expressing GATA-3, driven by the human CD2 locus control region (CD2-GATA3) [22], resulting in DO11.10xCD2-GATA3 mice. Mice used for experiments were backcrossed on a BALB/c background for a minimum of eight generations and used at an age of 8–12 wk. Mice were housed under specific pathogen-free conditions and all animal studies were performed according to institutional and state guidelines.\n\nIsolation of CD4+ T cells.\nCD4+ T cells were isolated from blood of healthy human volunteers using the anti-CD4 magnetic beads (Dynal, Hamburg, Germany) as previously described [60]. The purity of CD4+ T cells was initially tested by FACS and was ≥ 95%. Monoclonality of T cell clones was confirmed by TCR-chain mapping and was identified to be Vbeta8 positive. The clones were characterized by high IL-4 secretion.\n\nQuantitative real-time PCR.\nThe PCR primers and probes were designed based on the sequences reported in GenBank with the Primer Express software version 1.2 (Applied Biosystems) as follows: FOXP3 forward primer 5′-GAA ACA GCA CAT TCC CAG AGT TC-3′; FOXP3 reverse primer 5′-ATG GCC CAG CGG ATG AG-3′; EF-1α forward primer and reverse primer as described [61]; GATA3 forward primer 5′-GCG GGC TCT ATC ACA AAA TGA-3′ and rwd 5′-GCT CTC CTG GCT GCA GAC AGC-3′. The prepared cDNAs were amplified using SYBR-PCR mastermix (Biorad) according to the recommendations of the manufacturer in an ABI PRISM 7000 Sequence Detection System (Applied Biosystems).\nQuantitative PCR of murine samples was performed with Brilliant SYBR Green QPCR master mix (Stratagene) and the following primers: Ubiquitin C, 5′- AGG TCA AAC AGG AAG ACA GAC GTA-3′ and 5′-TCACACCCAAGAACAAGCACA-3′; Smad-7, 5′-GAA ACC GGG GGA ACG AAT TAT-3′ and 5′-CGC GAG TCT TCT CCT CCC A-3′; TGF-ß1, 5′-TGA CGT CAC TGG AGT TGT ACG G-3′ and 5′-GGT TCA TGT CAT GGA TGG TGC-3′. Primer pairs were evaluated for integrity by analysis of the amplification plot, dissociation curves, and efficiency of PCR amplification. PCR conditions were 10 min at 95 °C, followed by 40 cycles of 15 s at 95 °C and 60 °C for 1 min using an 7300 real-time PCR system (Applied Biosystems). PCR amplification of the housekeeping gene encoding ubiquitin C was performed during each run for each sample to allow normalization between samples. Relative quantification and calculation of the range of confidence was performed using the comparative ΔΔCT method.\n\nInducible murine Treg culture.\nNaive CD4+ T cells (CD4+, CD62L+, and CD25–) were isolated from pooled lymph nodes and spleens by FACS (FACS Aria, BD Biosciences). 5 × 105 T cells were co-cultured with 2.5 × 104 bone marrow–derived dendritic cells [62] and 0.01 μg/ml OVA323–339 peptide (Ansynth) in the presence or absence of 20-ng/μl rhTGF-ß1 (Peprotech) in 48-well plates. After 4 d, cells were harvested and analyzed for intracellular FOXP3 expression by FACS or gene expression by quantitative RT-PCR.\n\nIn vitro T cell differentiation.\nCD4+ CD45RA+ magnetically-sorted (CD45RO depletion, MACS, according to the protocol of the manufacturer) cells were stimulated with immobilized plate-bound anti-CD3 (1 μg/ml, Okt3, IgG1) and anti-CD28 (2 μg/ml) in Th1 conditions: 25 ng/ml IL-12, 5 μg/ml anti-IL-4 (R\u0026D systems); in Th2 conditions: 25 ng/ml IL-4, 5 μg/ml anti-IFN-γ, 5 μg/ml anti-IL-12 (R\u0026D systems); or in Treg conditions: 10 ng/ml TGF-β, 5 μg/ml anti-IFN-γ, 5 μg/ml anti-IL-12, 5 μg/ml anti-IL-4. Proliferating cells were expanded in medium containing IL-2 (30 ng/ml).\n\nCloning of the FOXP3 promoter and construction of mutant constructs.\nThe FOXP3 promoter was cloned into the pGL3 basic vector (Promega Biotech) to generate the pGL3 FOXP3 −511/+176 [24]. Site-directed mutagenesis in the FOXP3 promoter region were introduced using the QuickChange kit (Stratagene), according to the manufacturer's instructions. The following primer and its complementary strand were used: GTT TCT CAT GAG CCC TAT TAA GTC ATT CTT ACC TCT CAC CTC TGT GGT GA.\n\nTransfections and reporter gene assays.\nT cells were rested in serum-free AIM-V medium (Life Technologies) overnight. 3.5 μg of the FOXP3 promoter luciferase reporter vector and 0.5 μg phRL-TK were added to 3 × 106 CD4+ T cells resuspended in 100 μL of Nucleofector solution (Amaxa Biosystems) and electroporated using the U-15 program of the Nucleofector. After a 24-h culture in serum-free conditions and stimuli as indicated in the figures, luciferase activity was measured by the dual luciferase assay system (Promega Biotech) according to the manufacturer's instructions. Data were normalized by the activity of renilla luciferase.\n\nRNA isolation and cDNA synthesis.\nRNA was isolated using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. Reverse transcription of human samples was performed with TaqMan reverse-transcription reagents (Applied Biosystems) with random hexamers according to the manufacturer's protocol.\n\nRecombinant TAT proteins.\nThe cDNAs encoding GATA3 protein or the truncated GATA3 (lacking the two zinc fingers) were cloned in frame into an expression vector along with the TAT sequence as previously described [63]. Proteins were expressed in BL21 Star (DE3)pLysS (Invitrogen) and lysates were purified by Ni2+-chelate column chromatography. Both TAT-linked proteins were more than 95% pure, based on Coomassie blue staining of sodium disulfate acrylamide gels.\n\nTAT-GATA3 transduction.\nCD4+CD45RA+ cells were cultured in AIMV medium and transduced with 20 nM, 10 nM, or 500 nM of full-length or truncated GATA3 over the course of 4 h. After 4 h, the cells were washed and activated with soluble anti-CD3 and anti-CD28 and TGF-β (10 ng/ml). Each day, the TAT proteins were freshly added to the medium. FOXP3 expression was measured after 5 d by intracellular staining.\n\nIntracellular cytokine staining.\nT cells were stimulated with 2 × 10−7 M PMA and 1 μg/ml of ionomycin (Sigma Chemicals) for 4 h. The following mAb was used: anti-IL-4-PE (8D4–8, BD). Matched isotype controls were used at the same protein concentration as the respective antibodies. Four-color FACS was performed using an EPICS XL-MCL (Beckman Coulter) using the software Expo32 version for data acquisition and evaluation.\n\nFlow cytometry.\nFor analysis of FOXP3 expression at the single-cell level, cells were first stained with the monoclonal antibody CD25 (Beckman Coulter), and after fixation and permeabilization, cells were incubated with PE-conjugated monoclonal antibody PCH101 (anti-human FOXP3; eBioscience) based on the manufacturer's recommendations and subjected to FACS (EPICS XL-MCL). For cell surface marker staining, cells were incubated for 20 min at 4°C in staining buffer with the following antibodies: anti–CD152-PE (CTLA-4; BD), anti–PD-1 (eBiosciences), anti-GITR (R \u0026 D Systems), anti-CD69 (Beckman Coulter), anti-CD103 (DakoCytomation), anti-CD62L (Beckman Coulter), or anti-HLA-DR (Beckman Coulter). The controls were FITC, PE, or ECD-conjugated mouse IgG1 or rat IgG2a. For staining of mouse cells, the following mAbs from BD Biosciences were used following standard techniques as described above: anti-CD3, anti-CD4, and anti-CD25. Anti-FcγRII/III antibody (2.4G2, ATCC) was included in all stainings to reduce nonspecific antibody binding. To isolate naive murine CD4 T cells from murine DO11.10 or DO11.10xCD2-GATA3 T cells, cells were stained with anti-CD25-FITC, anti-CD62L-PE, and anti-CD4-APC prior to sorting. Dead cells were excluded with 4′',6-Diamidino-2-phenylindole (DAPI). To analyze murine Foxp3 expression in inducible Treg cultures, cells were stained intracellularly with anti-Foxp3-PE according to manufacturer's instruction, in conjunction with anti-CD4-APC and LIVE/DEAD fixable dead cell stain kit (Invitrogen) to discriminate live cells. All monoclonal antibodies for murine cell stainings were purchased from eBioscience or BD Biosciences.\n\nWestern blotting.\nFor FOXP3 analysis on the protein level, 1 × 106 CD4+CD25– cells were lysed and loaded next to a protein-mass ladder (Magicmark, Invitrogen) on a NuPAGE 4–12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Life Science). Unspecific binding was blocked with BSA, and the membranes were subsequently incubated with an 1:200 dilution of goat anti-FOXP3 in blocking buffer (Abcam) overnight at 4 °C. The blots were developed using an anti-goat HRP-labeled mAb (Amersham Biosciences) and visualized with a LAS 1000 camera (Fuji). Membranes were incubated in stripping buffer and re-blocked for 1 h. The membranes were re-probed using anti-GATA3 (HG3–31; Santa Cruz Biotechnology), anti-T-bet (4B10, Santa Cruz Biotechnology), anti-GAPDH (6C5, Ambion), anti-phospho-SMAD2 (138D4), anti-phospho-STAT6 (5A4), and anti-STAT6 (Cell Signaling Technology),\n\nAdministration of cytokines and antibodies in vivo.\nAge- and gender-matched normal B6 mice received every other day intraperitoneal (ip) injections of PBS, 1.5 μg rmIL-4, 50 μg anti-IL-4 mAb (11B11 or MAB404), or a mixture of 1.5 μg rmIL-4 plus 50 μg anti-IL-4 mAb (11B11 or MAB404) for 7 d. Thereafter, spleen and lymph node cells were analyzed by flow cytometry for CD3, CD4, and CD25 expression. The anti-mouse IL-4 mAb MAB404 was obtained from R\u0026D Systems, the second anti-mouse IL-4 mAb 11B11 was purchased from eBioscience.\n\nChIP.\nChIP analysis was performed according to the manufacturer's protocol (Upstate Biotechnology) with the following modifications. iTreg and Th2 cells were fixed with 1% formaldehyde for 10 min at room temperature. The chromatin was sheared to 200-1000 bp of length by sonication with five pulses of 10 s at 30% power (Bandelin). The chromatin was pre-cleared for 2 h with normal mouse IgG beads and then incubated with anti-GATA3-agarose beads (HG3–31; Santa Cruz Biotechnology) for 2 h. Washing and elution buffers were used according to the protocol of Upstate Biotechnology. Crosslinks were reversed by incubation at 65 °C for 4 h in the presence of 0.2 M NaCl, and the DNA was purified by phenol/chloroform extraction. The amount of DNA was determined by conventional PCR. The PCR addressed for the FOXP3 promoter region −246 to −511 and was performed using the following primers: 5′-gtgccctttacgagt catctg-3′ and 5′-gtgccctttacgagtcatctg-3′. The PCR products were visualized using an ethidium bromide gel.\n\nPull-down assay.\nCD4+ T cells were stimulated with PMA and ionomycin for 2 h at 37°C. The cells were pelleted, resuspended in buffer C (20 mM HEPES [pH 7.9], 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM DTT, protease inhibitors [Sigma]. and 0.1% NP-40) and lysed on ice for 15 min. Insoluble material was removed by centrifugation. The supernatant was diluted 1:3 with buffer D (as buffer C, but without NaCl). The lysates were incubated with 10 μg of poly(dI-dC) (Sigma) and 70 μl of streptavidin-agarose (Amersham Biosciences) carrying biotinylated oligonucleotides, for 3 h at 4 °C. The beads were washed twice with buffer C:D (1:3) and resuspended in DTT-containing loading buffer (NuPAGE; Invitrogen), heated to 70 °C for 10 min, and the eluants on a NuPAGE 4–12% bis-tris gel (Invitrogen). The proteins were electroblotted onto a PVDF membrane (Amersham Biosciences) and detected using an anti-GATA3 mAb (Santa Cruz Biotechnology). Accumulated signals were analyzed using AIDA software (Raytest).\n"}

GO-BP

GO-MF

GO-CC

sentences

events-check-again

bionlp-st-ge-2016-reference-tees

bionlp-st-ge-2016-reference

bionlp-st-ge-2016-uniprot

test2

PMC:2222968 / 35148-46702 JSONTXT

Annnotations TAB JSON ListView MergeView

PMC:2222968 / 35148-46702 JSON TXT