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  • 99
    Millipore ly364947
    Ly364947, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 71 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp tgfbr1 hs00610320 m1
    Gene Exp Tgfbr1 Hs00610320 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 98/100, based on 64 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore tgf β receptor i tgf βri kinase inhibitor
    <t>TGF-β1</t> is associated with curcumin-mediated generation of regulatory T cells at late phase. CD4 + T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (1∶10 for bead-to-cell ratio) or with 2 µg/mL of curcumin (Cur.) for 3 days, and then transferred to a new cell culture plate and incubated with a fresh media for an additional 3 days. (A) Cells were collected at the indicated time points and labeled with anti-CD25 and anti-Foxp3 antibodies. The cells were then washed and analyzed for CD25 and Foxp3 expression by flow cytometry. The numbers in each panel and the numbers in blankets indicate the percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells, respectively. (B) After 6 days of culture, cells were collected and percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells was determined by flow cytometric analysis. The empty and filled bars indicate cells treated with beads only and cells treated with both beads and curcumin, respectively. (C) A <t>TGF-βRI</t> kinase inhibitor (5 µg/mL) was added after 3 days of culture. The percentage of Foxp3 + cells was determined by flow cytometry. (D) After 3 days of culture, the cells were washed with PBS and then co-cultured with CFSE-labeled autologous CD4 + T cells with or without CD2/CD3/CD28 stimulation for 3 days. The cell proliferation was determined by flow cytometric analysis. (B, C and D) Data are presented as the mean ± SD. * P
    Tgf β Receptor I Tgf βri Kinase Inhibitor, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Selleck Chemicals tgf β receptor i kinase inhibitor
    <t>TGF-β1</t> is associated with curcumin-mediated generation of regulatory T cells at late phase. CD4 + T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (1∶10 for bead-to-cell ratio) or with 2 µg/mL of curcumin (Cur.) for 3 days, and then transferred to a new cell culture plate and incubated with a fresh media for an additional 3 days. (A) Cells were collected at the indicated time points and labeled with anti-CD25 and anti-Foxp3 antibodies. The cells were then washed and analyzed for CD25 and Foxp3 expression by flow cytometry. The numbers in each panel and the numbers in blankets indicate the percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells, respectively. (B) After 6 days of culture, cells were collected and percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells was determined by flow cytometric analysis. The empty and filled bars indicate cells treated with beads only and cells treated with both beads and curcumin, respectively. (C) A <t>TGF-βRI</t> kinase inhibitor (5 µg/mL) was added after 3 days of culture. The percentage of Foxp3 + cells was determined by flow cytometry. (D) After 3 days of culture, the cells were washed with PBS and then co-cultured with CFSE-labeled autologous CD4 + T cells with or without CD2/CD3/CD28 stimulation for 3 days. The cell proliferation was determined by flow cytometric analysis. (B, C and D) Data are presented as the mean ± SD. * P
    Tgf β Receptor I Kinase Inhibitor, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 95/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    77
    Scios Inc tgf βri kinase
    <t>TGF-β1</t> is associated with curcumin-mediated generation of regulatory T cells at late phase. CD4 + T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (1∶10 for bead-to-cell ratio) or with 2 µg/mL of curcumin (Cur.) for 3 days, and then transferred to a new cell culture plate and incubated with a fresh media for an additional 3 days. (A) Cells were collected at the indicated time points and labeled with anti-CD25 and anti-Foxp3 antibodies. The cells were then washed and analyzed for CD25 and Foxp3 expression by flow cytometry. The numbers in each panel and the numbers in blankets indicate the percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells, respectively. (B) After 6 days of culture, cells were collected and percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells was determined by flow cytometric analysis. The empty and filled bars indicate cells treated with beads only and cells treated with both beads and curcumin, respectively. (C) A <t>TGF-βRI</t> kinase inhibitor (5 µg/mL) was added after 3 days of culture. The percentage of Foxp3 + cells was determined by flow cytometry. (D) After 3 days of culture, the cells were washed with PBS and then co-cultured with CFSE-labeled autologous CD4 + T cells with or without CD2/CD3/CD28 stimulation for 3 days. The cell proliferation was determined by flow cytometric analysis. (B, C and D) Data are presented as the mean ± SD. * P
    Tgf βri Kinase, supplied by Scios Inc, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Millipore tβri kinase inhibitor sb431542
    <t>TGF-β1</t> is associated with curcumin-mediated generation of regulatory T cells at late phase. CD4 + T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (1∶10 for bead-to-cell ratio) or with 2 µg/mL of curcumin (Cur.) for 3 days, and then transferred to a new cell culture plate and incubated with a fresh media for an additional 3 days. (A) Cells were collected at the indicated time points and labeled with anti-CD25 and anti-Foxp3 antibodies. The cells were then washed and analyzed for CD25 and Foxp3 expression by flow cytometry. The numbers in each panel and the numbers in blankets indicate the percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells, respectively. (B) After 6 days of culture, cells were collected and percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells was determined by flow cytometric analysis. The empty and filled bars indicate cells treated with beads only and cells treated with both beads and curcumin, respectively. (C) A <t>TGF-βRI</t> kinase inhibitor (5 µg/mL) was added after 3 days of culture. The percentage of Foxp3 + cells was determined by flow cytometry. (D) After 3 days of culture, the cells were washed with PBS and then co-cultured with CFSE-labeled autologous CD4 + T cells with or without CD2/CD3/CD28 stimulation for 3 days. The cell proliferation was determined by flow cytometric analysis. (B, C and D) Data are presented as the mean ± SD. * P
    Tβri Kinase Inhibitor Sb431542, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore tβri kinase inhibitor sb505124
    NUAK1 and NUAK2 are transcriptionally induced by TGFβ. A , real-time qRT-PCR analysis of NUAK1 mRNA normalized to HPRT1 mRNA from AG1523 cells after treatment with cycloheximide (20 μ m ) or an equivalent volume of PBS as negative control for 1 h followed by TGFβ (1 ng/ml) stimulation for 5 h. On the right , corresponding immunoblot for NUAK1, phospho-SMAD2 and total SMAD2 under the conditions used in the samples for real-time qRT-PCR. Molecular size markers in kDa are shown. One representative experiment of two is shown. B , real-time qRT-PCR analysis of Nuak2 mRNA normalized to Gapdh mRNA from NMuMG cells after treatment with vehicle or cycloheximide for 15 min followed by TGFβ (5 ng/ml) stimulation for 1 h. Immunoblots of NUAK2, phospho-SMAD2, and total SMAD2 proteins serve as controls for the RNA analysis. SMAD2 serves as a protein-loading control. Molecular size markers in kDa are shown. C , NMuMG cells were pretreated with vehicle or actinomycin D for 1 h before treatment with TGFβ (5 ng/ml) for 1 h. Nuak2 mRNA was normalized to Gapdh mRNA as measured by real-time qRT-PCR. D , immunoblotting of NUAK1 and SMAD4. AG1523 cells were stimulated with TGFβ (1 ng/ml) for 5 h. Ponceau-S staining of the immunoblot serves as a protein-loading control. Molecular size markers in kDa are shown. E , mRNA expression of Nuak2 , Gadd45 γ, and Smad4 in NMuMG cells after treatment with control or Smad4 siRNA and TGFβ (5 ng/ml) stimulation for 1 h. F , immunoblots of NUAK2, PAI-1, SMAD4, and β-actin proteins in MDA-MB-468 cells transiently infected with Adex-LacZ or Adex-SMAD4 (the latter at two different multiplicities of infection, 1 and 4) prior to cell starvation and stimulation with TGFβ (5 ng/ml) for 24 h. β-Actin serves as protein-loading control; a star shows nonspecific protein bands. Molecular size markers in kDa are shown. G , immunoblotting of NUAK1 and NUAK2 in AG1523 cells after treatment with TGFβ (5 ng/ml) for 3 h in the presence of <t>TβRI</t> kinase inhibitors LY2157299 (5 μ m ) or <t>SB505124</t> (2.5 μ m ) or DMSO (0.1%). Molecular size markers in kDa are shown. H , immunoblots of NUAK2, phospho-ERK1/2, phospho-SMAD2, phospho-SMAD3, SMAD4, and β-actin proteins in NMuMG cells serum-starved overnight, followed by pretreatment with the indicated inhibitors or vehicle (DMSO) for 1 h prior to stimulation with TGFβ (1 ng/ml) for 6 h. β-Actin serves as protein-loading control. Molecular size markers in kDa are shown. Data are presented as mean ± S.E. ( error bars ) after performing at least three independent experiments. Asterisks imply significant differences compared with controls: *, p
    Tβri Kinase Inhibitor Sb505124, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cayman Chemical tβri kinase inhibitor ly2157299
    NUAK1 and NUAK2 are transcriptionally induced by TGFβ. A , real-time qRT-PCR analysis of NUAK1 mRNA normalized to HPRT1 mRNA from AG1523 cells after treatment with cycloheximide (20 μ m ) or an equivalent volume of PBS as negative control for 1 h followed by TGFβ (1 ng/ml) stimulation for 5 h. On the right , corresponding immunoblot for NUAK1, phospho-SMAD2 and total SMAD2 under the conditions used in the samples for real-time qRT-PCR. Molecular size markers in kDa are shown. One representative experiment of two is shown. B , real-time qRT-PCR analysis of Nuak2 mRNA normalized to Gapdh mRNA from NMuMG cells after treatment with vehicle or cycloheximide for 15 min followed by TGFβ (5 ng/ml) stimulation for 1 h. Immunoblots of NUAK2, phospho-SMAD2, and total SMAD2 proteins serve as controls for the RNA analysis. SMAD2 serves as a protein-loading control. Molecular size markers in kDa are shown. C , NMuMG cells were pretreated with vehicle or actinomycin D for 1 h before treatment with TGFβ (5 ng/ml) for 1 h. Nuak2 mRNA was normalized to Gapdh mRNA as measured by real-time qRT-PCR. D , immunoblotting of NUAK1 and SMAD4. AG1523 cells were stimulated with TGFβ (1 ng/ml) for 5 h. Ponceau-S staining of the immunoblot serves as a protein-loading control. Molecular size markers in kDa are shown. E , mRNA expression of Nuak2 , Gadd45 γ, and Smad4 in NMuMG cells after treatment with control or Smad4 siRNA and TGFβ (5 ng/ml) stimulation for 1 h. F , immunoblots of NUAK2, PAI-1, SMAD4, and β-actin proteins in MDA-MB-468 cells transiently infected with Adex-LacZ or Adex-SMAD4 (the latter at two different multiplicities of infection, 1 and 4) prior to cell starvation and stimulation with TGFβ (5 ng/ml) for 24 h. β-Actin serves as protein-loading control; a star shows nonspecific protein bands. Molecular size markers in kDa are shown. G , immunoblotting of NUAK1 and NUAK2 in AG1523 cells after treatment with TGFβ (5 ng/ml) for 3 h in the presence of <t>TβRI</t> kinase inhibitors <t>LY2157299</t> (5 μ m ) or SB505124 (2.5 μ m ) or DMSO (0.1%). Molecular size markers in kDa are shown. H , immunoblots of NUAK2, phospho-ERK1/2, phospho-SMAD2, phospho-SMAD3, SMAD4, and β-actin proteins in NMuMG cells serum-starved overnight, followed by pretreatment with the indicated inhibitors or vehicle (DMSO) for 1 h prior to stimulation with TGFβ (1 ng/ml) for 6 h. β-Actin serves as protein-loading control. Molecular size markers in kDa are shown. Data are presented as mean ± S.E. ( error bars ) after performing at least three independent experiments. Asterisks imply significant differences compared with controls: *, p
    Tβri Kinase Inhibitor Ly2157299, supplied by Cayman Chemical, used in various techniques. Bioz Stars score: 82/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Epichem tgf β receptor i kinase inhibitor
    NUAK1 and NUAK2 are transcriptionally induced by TGFβ. A , real-time qRT-PCR analysis of NUAK1 mRNA normalized to HPRT1 mRNA from AG1523 cells after treatment with cycloheximide (20 μ m ) or an equivalent volume of PBS as negative control for 1 h followed by TGFβ (1 ng/ml) stimulation for 5 h. On the right , corresponding immunoblot for NUAK1, phospho-SMAD2 and total SMAD2 under the conditions used in the samples for real-time qRT-PCR. Molecular size markers in kDa are shown. One representative experiment of two is shown. B , real-time qRT-PCR analysis of Nuak2 mRNA normalized to Gapdh mRNA from NMuMG cells after treatment with vehicle or cycloheximide for 15 min followed by TGFβ (5 ng/ml) stimulation for 1 h. Immunoblots of NUAK2, phospho-SMAD2, and total SMAD2 proteins serve as controls for the RNA analysis. SMAD2 serves as a protein-loading control. Molecular size markers in kDa are shown. C , NMuMG cells were pretreated with vehicle or actinomycin D for 1 h before treatment with TGFβ (5 ng/ml) for 1 h. Nuak2 mRNA was normalized to Gapdh mRNA as measured by real-time qRT-PCR. D , immunoblotting of NUAK1 and SMAD4. AG1523 cells were stimulated with TGFβ (1 ng/ml) for 5 h. Ponceau-S staining of the immunoblot serves as a protein-loading control. Molecular size markers in kDa are shown. E , mRNA expression of Nuak2 , Gadd45 γ, and Smad4 in NMuMG cells after treatment with control or Smad4 siRNA and TGFβ (5 ng/ml) stimulation for 1 h. F , immunoblots of NUAK2, PAI-1, SMAD4, and β-actin proteins in MDA-MB-468 cells transiently infected with Adex-LacZ or Adex-SMAD4 (the latter at two different multiplicities of infection, 1 and 4) prior to cell starvation and stimulation with TGFβ (5 ng/ml) for 24 h. β-Actin serves as protein-loading control; a star shows nonspecific protein bands. Molecular size markers in kDa are shown. G , immunoblotting of NUAK1 and NUAK2 in AG1523 cells after treatment with TGFβ (5 ng/ml) for 3 h in the presence of <t>TβRI</t> kinase inhibitors <t>LY2157299</t> (5 μ m ) or SB505124 (2.5 μ m ) or DMSO (0.1%). Molecular size markers in kDa are shown. H , immunoblots of NUAK2, phospho-ERK1/2, phospho-SMAD2, phospho-SMAD3, SMAD4, and β-actin proteins in NMuMG cells serum-starved overnight, followed by pretreatment with the indicated inhibitors or vehicle (DMSO) for 1 h prior to stimulation with TGFβ (1 ng/ml) for 6 h. β-Actin serves as protein-loading control. Molecular size markers in kDa are shown. Data are presented as mean ± S.E. ( error bars ) after performing at least three independent experiments. Asterisks imply significant differences compared with controls: *, p
    Tgf β Receptor I Kinase Inhibitor, supplied by Epichem, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Promega tβri kinase
    GPR50 inhibits cell proliferation and tumor growth in MDA-MB-231 cells. a Expression of GPR50Δ4 and GPR50wt in lysates of MDA-MB-231 cell pools revealed by western blot. b An equal number of MDA-MB-231 cells expressing Mock, GPR50Δ4 or GPR50wt were seeded into 96-well plates, starved and stimulated with TGFβ (2 ng/mL) and transfected with either siRNA against control (si-Ctrl) or <t>TβRI</t> (si-TβRI). The proliferation rate over total amount of cells was measured with the MTT assay 5 days after starvation (Mean ± s.e.m., n = 5 independent experiments, * p
    Tβri Kinase, supplied by Promega, used in various techniques. Bioz Stars score: 95/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris tgf βri
    Albumin-induced increase in matrix metalloproteinase (MMP)-9 occurs independently of the transforming growth factor <t>(TGF)-β</t> receptor. Representative zymogram and corresponding quantification of the level of MMP-9 release by astrocytes treated with BSA in the presence (+) or absence (−) of (a) the TGF-β receptor I inhibitor <t>SB431542,</t> or (b) the Smad pathway inhibitor SIS3; neither inhibition of the TGF-β receptor I inhibitor nor the Smad pathway inhibited albumin-induced increase in MMP-9. (c) Representative zymogram of the level of MMP-9 release by astrocytes treated with BSA and TGF-β1 (10 ng/mL). Data are representative of mean ± SEM of three independent experiments. *** P
    Tgf βri, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Millipore tβri kinase inhibitor ii
    Increased expression of <t>TβRI</t> and TβRII in CD4 + CD25 – T cells of PARP-1 −/− mice. (A-C) Quantitative PCR analysis of the expression of Tgfbr1 and Tgfbr2 mRNA in (A) freshly isolated splenic CD4 + CD25 - T cells from PARP-1
    Tβri Kinase Inhibitor Ii, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Scios Inc tgf β receptor i tgfβri kinase inhibitor
    Increased expression of <t>TβRI</t> and TβRII in CD4 + CD25 – T cells of PARP-1 −/− mice. (A-C) Quantitative PCR analysis of the expression of Tgfbr1 and Tgfbr2 mRNA in (A) freshly isolated splenic CD4 + CD25 - T cells from PARP-1
    Tgf β Receptor I Tgfβri Kinase Inhibitor, supplied by Scios Inc, used in various techniques. Bioz Stars score: 78/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    84
    Tocris tβri kinase inhibitor
    Increased expression of <t>TβRI</t> and TβRII in CD4 + CD25 – T cells of PARP-1 −/− mice. (A-C) Quantitative PCR analysis of the expression of Tgfbr1 and Tgfbr2 mRNA in (A) freshly isolated splenic CD4 + CD25 - T cells from PARP-1
    Tβri Kinase Inhibitor, supplied by Tocris, used in various techniques. Bioz Stars score: 84/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eli Lilly tβri kinase inhibitors ly2109761
    Increased expression of <t>TβRI</t> and TβRII in CD4 + CD25 – T cells of PARP-1 −/− mice. (A-C) Quantitative PCR analysis of the expression of Tgfbr1 and Tgfbr2 mRNA in (A) freshly isolated splenic CD4 + CD25 - T cells from PARP-1
    Tβri Kinase Inhibitors Ly2109761, supplied by Eli Lilly, used in various techniques. Bioz Stars score: 79/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Scios Inc tgf β receptor i kinase inhibitor sd 208
    GDF-15 effects on the canonical <t>TGF-β</t> signaling pathway in glioma cells A. LNT-229 or LN-308 cells were exposed to GDF-15 (100 ng/ml), BMP-4 (5 ng/ml) or TGF-β 2 (10 ng/ml) as indicated for 24 h, harvested and assessed for pSmad2, Smad2/3, pSmad1/5/8 and Smad5 levels by immunoblot. B. LNT-229 or LN-308 cells transfected with the pGL3-SBE4-Luc reporter plasmid were exposed to TGF-β 2 , GDF-15, <t>SD-208</t> or combinations thereof as indicated for 24 h, and analyzed for firefly/renilla luciferase activity. C. LNT-229 and LN-308 cells transfected with the pGL3-SBE4-Luc reporter plasmid were pre-exposed to GDF-15 for 1.5 h or not as indicated, subsequently treated with TGF-β 2 for 24 h and analyzed as in (B) (* p
    Tgf β Receptor I Kinase Inhibitor Sd 208, supplied by Scios Inc, used in various techniques. Bioz Stars score: 80/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris tgf βri kinase activity inhibitor a83 01
    miR-27a directly targets <t>TGF-βRI</t> and represses the TGF-β signaling. a qRT-PCR analysis of TGF-βRI and TGF-βRII expression in cervical cancer cell lines versus normal cervical epithelia. b qRT-PCR. TGF-βRI expression was downregulated by miR-27a agomir in HeLa, but not in SiHa. c qRT-PCR. TGF-βRII levels in three cervical cancer cell lines were not affected by agomir transfection. d Results of double-luciferase reporter assays show the luciferase activity of the wild type TGF-βRI 3′-UTR (TGF-βRI-WT) and the mutant one (TGF-βRI-Mut) in the absence or presence of hsp-miR-27a-3p mimics. e – h qRT-PCR. Expression levels of TGF-βRI, SMAD2, SMAD3, and SMAD4 in cervical cancer cells that were transfected with agomir NC or miR-27a agomir. i Western blot. TGF-βRI, SMAD3, and p-SMAD3 were downregulated by miR-27a agomir transfection. ** P
    Tgf βri Kinase Activity Inhibitor A83 01, supplied by Tocris, used in various techniques. Bioz Stars score: 99/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore tβri kinase inhibitor sb4315242
    miR-27a directly targets <t>TGF-βRI</t> and represses the TGF-β signaling. a qRT-PCR analysis of TGF-βRI and TGF-βRII expression in cervical cancer cell lines versus normal cervical epithelia. b qRT-PCR. TGF-βRI expression was downregulated by miR-27a agomir in HeLa, but not in SiHa. c qRT-PCR. TGF-βRII levels in three cervical cancer cell lines were not affected by agomir transfection. d Results of double-luciferase reporter assays show the luciferase activity of the wild type TGF-βRI 3′-UTR (TGF-βRI-WT) and the mutant one (TGF-βRI-Mut) in the absence or presence of hsp-miR-27a-3p mimics. e – h qRT-PCR. Expression levels of TGF-βRI, SMAD2, SMAD3, and SMAD4 in cervical cancer cells that were transfected with agomir NC or miR-27a agomir. i Western blot. TGF-βRI, SMAD3, and p-SMAD3 were downregulated by miR-27a agomir transfection. ** P
    Tβri Kinase Inhibitor Sb4315242, supplied by Millipore, used in various techniques. Bioz Stars score: 81/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Tocris tβri kinase inhibitor sb431542
    TGF-β signaling is required for COL1 expression in human skin fibroblasts. a Fibroblasts were treated with vehicle ( Ctrl ) or <t>TβRI</t> inhibitor <t>SB431542</t> (10 μM) and analyzed 48 h after treatment. COL1A1 mRNA ( left panel
    Tβri Kinase Inhibitor Sb431542, supplied by Tocris, used in various techniques. Bioz Stars score: 77/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Induction of integrin αvβ6 expression requires <t>TGF-β</t> signaling and viral DNA replication A: Infected HUVECs were cultured with or without chicken anti-TGF-β polyclonal antibody (20 μg/ml), chicken IgY isotype control antibody (control Ab, 20 μg/ml), the <t>ALK5</t> kinase inhibitor <t>SB431542</t> (0.5 μmol/L), or the vehicle DMSO for 7 days, and surface expression of integrin αvβ6 was analyzed by flow cytometric analysis. Typical histograms are shown. Shaded areas represent expression of specific proteins. Lines represent isotype control. Experiments were repeated at least two times. B: Surface expression of integrin αvβ6 was analyzed by flow cytometric analysis at 7 days after infection with or without viral DNA polymerase inhibitors, Foscarnet, and phosphonoacetic acid (PAA). Typical histograms are shown. Shaded areas represent expression of specific proteins. Lines represent isotype control. Experiments were repeated six times. C: Active TGF-β was not produced by infected HUVECs in the presence of viral DNA polymerase inhibitors. Equal numbers of TMLC TGF-β reporter cells and control (cont.) or infected HUVECs were cultured for 16 to 24 hours at 7 days after infection. Relative luciferase activity in cell lysates was defined as the measured activity divided by TMLC baseline activity. Representative data (mean ± SE) are from four experiments done in triplicate.
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    TGF-β signaling pathway. Bioactive TGF-β binds to the TGF-β type II receptor (TβRII), recruiting the TGF-β type I receptor <t>(TβRI</t> a.k.a. <t>ALK5).</t> TβRII phosphorylates and activates TβRI/ALK5,
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    TGF-β signaling pathway. Bioactive TGF-β binds to the TGF-β type II receptor (TβRII), recruiting the TGF-β type I receptor <t>(TβRI</t> a.k.a. <t>ALK5).</t> TβRII phosphorylates and activates TβRI/ALK5,
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    Generation of a novel nondiffusible <t>TβRI</t> kinase inhibitor. ( A ) Structure of pyrogallol (Pg), 3Abd, and derivatives. ( B ) A549 cells were stimulated with TGF-β1 for 30 minutes and lysates immunoblotted for p-Smad3, Smad3, and β-actin. Treatment without preincubation: 3Abd (0.5–10 μM); 2Abd or 4Abd (1, 10 μM). ( C ) A549 cells were stimulated with TGF-β1 for 48 hours and lysates immunoblotted for LOXL2, fibronectin, E-cadherin, Snail1, and β-actin. 3Abd was added at concentraions ranging from 0.5 to 10 μM. ( D ) Purified ALK5/TβRI catalytic domain kinase assay was performed with 10 doses of 3Abd or Pg starting from 100 μM. Kinase activity was indicated by 33 P-ATP signals, and IC 50 of 3Abd was calculated as approximately 3 μM. B – D are representative of 3 experiments with similar results. ( E ) SMAD-binding element (SBE) reporter–transfected NMuMG and A549 cells were seeded into a 96-well plate. Cocultured wells were seeded with 5,000 transfected NMuMG cells and 25,000 nontransfected A549 cells. Cells were pretreated with or without 1 μM corilagin, 1 μM EGCG, 10 μM 3Abd, or 5 μM SB431542 for 6 hours and stimulated with TGF-β1 overnight before lysis for luciferase assay. Data are presented as percent TGF-β1–induced SBE luciferase activity of DMSO control in log scale. Mean ± SD, n = 3. ( F ) Flag-tagged TβRI and TβRII were immunoprecipitated from A549 cells and in vitro kinase assay performed on beads exposed to lysate pretreated with corilagin or DMSO. The final reaction was eluted and analyzed by immunoblotting for phosphotyrosine and TβRI. The phosphotyrosine bands were quantified using ImageJ and normalized to DMSO control. Data represent mean ± SD. P value by unpaired 2-tailed t test of 6 separate experiments. ( G ) Schematic overview of mechanism. A trihydroxyphenolic-containing compound engages active LOXL2, initiating auto-oxidation of K731 and creating a key allysine inactivating the enzyme. In the process, a 3Abd-like metabolite is generated that then blocks TβRI kinase. The combined effects block pathological collagen accumulation.
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    Generation of a novel nondiffusible <t>TβRI</t> kinase inhibitor. ( A ) Structure of pyrogallol (Pg), 3Abd, and derivatives. ( B ) A549 cells were stimulated with TGF-β1 for 30 minutes and lysates immunoblotted for p-Smad3, Smad3, and β-actin. Treatment without preincubation: 3Abd (0.5–10 μM); 2Abd or 4Abd (1, 10 μM). ( C ) A549 cells were stimulated with TGF-β1 for 48 hours and lysates immunoblotted for LOXL2, fibronectin, E-cadherin, Snail1, and β-actin. 3Abd was added at concentraions ranging from 0.5 to 10 μM. ( D ) Purified ALK5/TβRI catalytic domain kinase assay was performed with 10 doses of 3Abd or Pg starting from 100 μM. Kinase activity was indicated by 33 P-ATP signals, and IC 50 of 3Abd was calculated as approximately 3 μM. B – D are representative of 3 experiments with similar results. ( E ) SMAD-binding element (SBE) reporter–transfected NMuMG and A549 cells were seeded into a 96-well plate. Cocultured wells were seeded with 5,000 transfected NMuMG cells and 25,000 nontransfected A549 cells. Cells were pretreated with or without 1 μM corilagin, 1 μM EGCG, 10 μM 3Abd, or 5 μM SB431542 for 6 hours and stimulated with TGF-β1 overnight before lysis for luciferase assay. Data are presented as percent TGF-β1–induced SBE luciferase activity of DMSO control in log scale. Mean ± SD, n = 3. ( F ) Flag-tagged TβRI and TβRII were immunoprecipitated from A549 cells and in vitro kinase assay performed on beads exposed to lysate pretreated with corilagin or DMSO. The final reaction was eluted and analyzed by immunoblotting for phosphotyrosine and TβRI. The phosphotyrosine bands were quantified using ImageJ and normalized to DMSO control. Data represent mean ± SD. P value by unpaired 2-tailed t test of 6 separate experiments. ( G ) Schematic overview of mechanism. A trihydroxyphenolic-containing compound engages active LOXL2, initiating auto-oxidation of K731 and creating a key allysine inactivating the enzyme. In the process, a 3Abd-like metabolite is generated that then blocks TβRI kinase. The combined effects block pathological collagen accumulation.
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    Galunisertib activities in 13 PDX samples and its correlations with <t>TGF-βRI</t> mRNA and pSMAD2 protein expression. ( a ) The in vivo efficacies were evaluated by ABC (see materials and methods ) values and compared to TGF-βRI mRNA expression (Affymetrix HGU133 Plus 2.0) and pSMAD2 protein expression (Western blotting; see Fig. 4 ) levels. ( b ) Correlation between TGF-βRI mRNA expression levels and ABC values (%). ( c ) Correlation between pSMAD2 protein expression levels and ABC values. P values ≤ 0.05 are considered significant
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    Galunisertib activities in 13 PDX samples and its correlations with <t>TGF-βRI</t> mRNA and pSMAD2 protein expression. ( a ) The in vivo efficacies were evaluated by ABC (see materials and methods ) values and compared to TGF-βRI mRNA expression (Affymetrix HGU133 Plus 2.0) and pSMAD2 protein expression (Western blotting; see Fig. 4 ) levels. ( b ) Correlation between TGF-βRI mRNA expression levels and ABC values (%). ( c ) Correlation between pSMAD2 protein expression levels and ABC values. P values ≤ 0.05 are considered significant
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    Morphometric analyses of mechanisms and pathways in rAAV-transduced chondrocytes in situ . Explants were transduced with rAAV- lacZ or rAAV-hTGF-β as described in Figure 1 and maintained in culture for up to 90 days. The samples were then fixed and histologically processed at the denoted time points to monitor the % of cells immunostained for MMP-13 (A) , TIMP-1 (B) , TIMP-3 (C) , PTHrP (D) , β-catenin (E) , and the <t>TGF-β</t> receptor I (ALK1 and ALK5) (F and G , respectively ) . Data on the ALK1/ALK5 ratio are presented in (H) .
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    Morphometric analyses of mechanisms and pathways in rAAV-transduced chondrocytes in situ . Explants were transduced with rAAV- lacZ or rAAV-hTGF-β as described in Figure 1 and maintained in culture for up to 90 days. The samples were then fixed and histologically processed at the denoted time points to monitor the % of cells immunostained for MMP-13 (A) , TIMP-1 (B) , TIMP-3 (C) , PTHrP (D) , β-catenin (E) , and the <t>TGF-β</t> receptor I (ALK1 and ALK5) (F and G , respectively ) . Data on the ALK1/ALK5 ratio are presented in (H) .
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    OvCa‐secreted <t>TGF‐β</t> transforms the pre‐metastatic peritoneum, favouring tumour progression. (A) Representative images of in vivo monitoring of SKOV3‐luc‐D3 cells in mice pre‐conditioned with TGF‐β1‐encoding adenovirus or control. Quantification of bioluminescence showed that tumour growth was increased in mice pre‐conditioned with TGF‐β1 adenovirus ( n = 6) as compared with control adenoviral pretreatment ( n = 6). (B) (a) Representative images of E‐cadherin immunostaining in the mesothelial monolayer of a mouse killed 2 days after being pre‐conditioned with conditioned medium (CM) from OvCa cells or control medium. Scale bars: 25 µm. (b) Diagram of experimental design. (c) Representative images of in vivo monitoring of SKOV3‐luc‐D3 cells and quantification of bioluminescence showed that intraperitoneal tumour growth was higher in mice pretreated with SKOV3 medium (CM). The TGF‐β receptor I inhibitor (GW) reduced tumour growth to levels comparable to those of mice whose peritoneums had not been pre‐conditioned (control medium). n = 6 per group. All mice were monitored for 41 days. Graphs represent mean average radiance (expressed as photons/s/cm 2 /sr) of SKOV3‐luc‐D3 cells ± standard error of the mean. Symbols represent the statistical differences over time between groups (** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). dpi, days post‐inoculation; i.p., intraperitoneal.
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    OvCa‐secreted <t>TGF‐β</t> transforms the pre‐metastatic peritoneum, favouring tumour progression. (A) Representative images of in vivo monitoring of SKOV3‐luc‐D3 cells in mice pre‐conditioned with TGF‐β1‐encoding adenovirus or control. Quantification of bioluminescence showed that tumour growth was increased in mice pre‐conditioned with TGF‐β1 adenovirus ( n = 6) as compared with control adenoviral pretreatment ( n = 6). (B) (a) Representative images of E‐cadherin immunostaining in the mesothelial monolayer of a mouse killed 2 days after being pre‐conditioned with conditioned medium (CM) from OvCa cells or control medium. Scale bars: 25 µm. (b) Diagram of experimental design. (c) Representative images of in vivo monitoring of SKOV3‐luc‐D3 cells and quantification of bioluminescence showed that intraperitoneal tumour growth was higher in mice pretreated with SKOV3 medium (CM). The TGF‐β receptor I inhibitor (GW) reduced tumour growth to levels comparable to those of mice whose peritoneums had not been pre‐conditioned (control medium). n = 6 per group. All mice were monitored for 41 days. Graphs represent mean average radiance (expressed as photons/s/cm 2 /sr) of SKOV3‐luc‐D3 cells ± standard error of the mean. Symbols represent the statistical differences over time between groups (** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). dpi, days post‐inoculation; i.p., intraperitoneal.
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    Differentiation of Th9 IL-4+IL-1β cells is transforming growth factor <t>(TGF)-β</t> independent. a – f T cells differentiation and gene array data are the same as shown in Fig. 2 . a Gene set enrichment analysis (GSEA) of Th9 IL-4+IL-1β cells versus classic Th9 IL-4+TGF-β cells or the Rest T helper (Th) cells for interleukin (IL)-1 signaling genes. Rest Th cells contain Th1, Th2, Th9 IL-4+TGF-β , and Th9 IL-4+TGF-β+IL-1β cells. b GSEA of Th9 IL-4+IL-1β versus classic Th9 IL-4+TGF-β cells or the Rest Th cells for TGF-β signaling genes. c , d Reverse transcriptase–PCR (RT-PCR) analysis of Il9 transcriptional level ( c ) and enzyme-linked immunosorbent assay (ELISA) of IL-9 production in the supernatants ( d ) from <t>TGF-βRi-treated</t> classic Th9 IL-4+TGF-β cells or Th9 IL-4+IL-1β cells at the indicated concentrations ( n = 3). TGF-βRi <t>TGF-β</t> receptor serine kinase inhibitor. e , f RT-PCR analysis of Il9 transcriptional level ( e ) and ELISA of IL-9 production in the supernatants ( f ) from αTGF-β-treated classic Th9 IL-4+TGF-β cells or Th9 IL-4+IL-1β cells at the indicated concentrations ( n = 3). αTGF-β TGF-β monoclonal antibody (mAb). g , h Naive CD4 + CD62L + T cells were purified from the spleens of wild-type (WT) mice or CD4dnTGF-βRII mice and cultured with plate-bound anti-CD3 mAbs and soluble anti-CD28 mAbs under polarized conditions, as detailed in the Methods section, for 3 days. RT-PCR analysis of Il9 transcriptional level ( g ) and ELISA of IL-9 production in the supernatants ( h ) of classic Th9 IL-4+TGF-β cells and Th9 IL-4+IL-1β cells from WT mice or CD4dnTGF-βRII mice after in vitro differentiation ( n = 3). CD4dnTGF-βRII mice mice expressing a dominant-negative TGF-β receptor. Data are mean ± SD; ** P
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    Differentiation of Th9 IL-4+IL-1β cells is transforming growth factor <t>(TGF)-β</t> independent. a – f T cells differentiation and gene array data are the same as shown in Fig. 2 . a Gene set enrichment analysis (GSEA) of Th9 IL-4+IL-1β cells versus classic Th9 IL-4+TGF-β cells or the Rest T helper (Th) cells for interleukin (IL)-1 signaling genes. Rest Th cells contain Th1, Th2, Th9 IL-4+TGF-β , and Th9 IL-4+TGF-β+IL-1β cells. b GSEA of Th9 IL-4+IL-1β versus classic Th9 IL-4+TGF-β cells or the Rest Th cells for TGF-β signaling genes. c , d Reverse transcriptase–PCR (RT-PCR) analysis of Il9 transcriptional level ( c ) and enzyme-linked immunosorbent assay (ELISA) of IL-9 production in the supernatants ( d ) from <t>TGF-βRi-treated</t> classic Th9 IL-4+TGF-β cells or Th9 IL-4+IL-1β cells at the indicated concentrations ( n = 3). TGF-βRi <t>TGF-β</t> receptor serine kinase inhibitor. e , f RT-PCR analysis of Il9 transcriptional level ( e ) and ELISA of IL-9 production in the supernatants ( f ) from αTGF-β-treated classic Th9 IL-4+TGF-β cells or Th9 IL-4+IL-1β cells at the indicated concentrations ( n = 3). αTGF-β TGF-β monoclonal antibody (mAb). g , h Naive CD4 + CD62L + T cells were purified from the spleens of wild-type (WT) mice or CD4dnTGF-βRII mice and cultured with plate-bound anti-CD3 mAbs and soluble anti-CD28 mAbs under polarized conditions, as detailed in the Methods section, for 3 days. RT-PCR analysis of Il9 transcriptional level ( g ) and ELISA of IL-9 production in the supernatants ( h ) of classic Th9 IL-4+TGF-β cells and Th9 IL-4+IL-1β cells from WT mice or CD4dnTGF-βRII mice after in vitro differentiation ( n = 3). CD4dnTGF-βRII mice mice expressing a dominant-negative TGF-β receptor. Data are mean ± SD; ** P
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    Image Search Results


    TGF-β1 is associated with curcumin-mediated generation of regulatory T cells at late phase. CD4 + T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (1∶10 for bead-to-cell ratio) or with 2 µg/mL of curcumin (Cur.) for 3 days, and then transferred to a new cell culture plate and incubated with a fresh media for an additional 3 days. (A) Cells were collected at the indicated time points and labeled with anti-CD25 and anti-Foxp3 antibodies. The cells were then washed and analyzed for CD25 and Foxp3 expression by flow cytometry. The numbers in each panel and the numbers in blankets indicate the percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells, respectively. (B) After 6 days of culture, cells were collected and percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells was determined by flow cytometric analysis. The empty and filled bars indicate cells treated with beads only and cells treated with both beads and curcumin, respectively. (C) A TGF-βRI kinase inhibitor (5 µg/mL) was added after 3 days of culture. The percentage of Foxp3 + cells was determined by flow cytometry. (D) After 3 days of culture, the cells were washed with PBS and then co-cultured with CFSE-labeled autologous CD4 + T cells with or without CD2/CD3/CD28 stimulation for 3 days. The cell proliferation was determined by flow cytometric analysis. (B, C and D) Data are presented as the mean ± SD. * P

    Journal: PLoS ONE

    Article Title: Curcumin Inhibits CD4+ T Cell Activation, but Augments CD69 Expression and TGF-?1-Mediated Generation of Regulatory T Cells at Late Phase

    doi: 10.1371/journal.pone.0062300

    Figure Lengend Snippet: TGF-β1 is associated with curcumin-mediated generation of regulatory T cells at late phase. CD4 + T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (1∶10 for bead-to-cell ratio) or with 2 µg/mL of curcumin (Cur.) for 3 days, and then transferred to a new cell culture plate and incubated with a fresh media for an additional 3 days. (A) Cells were collected at the indicated time points and labeled with anti-CD25 and anti-Foxp3 antibodies. The cells were then washed and analyzed for CD25 and Foxp3 expression by flow cytometry. The numbers in each panel and the numbers in blankets indicate the percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells, respectively. (B) After 6 days of culture, cells were collected and percentage of CD25 hi Foxp3 + cells in total and among CD25 + cells was determined by flow cytometric analysis. The empty and filled bars indicate cells treated with beads only and cells treated with both beads and curcumin, respectively. (C) A TGF-βRI kinase inhibitor (5 µg/mL) was added after 3 days of culture. The percentage of Foxp3 + cells was determined by flow cytometry. (D) After 3 days of culture, the cells were washed with PBS and then co-cultured with CFSE-labeled autologous CD4 + T cells with or without CD2/CD3/CD28 stimulation for 3 days. The cell proliferation was determined by flow cytometric analysis. (B, C and D) Data are presented as the mean ± SD. * P

    Article Snippet: U0126 (ERK inhibitor), SP600125 (JNK inhibitor), SB203580 (p38 MAPK inhibitor) and a TGF-β receptor I (TGF-βRI) kinase inhibitor were obtained from Calbiochem (San Diego, CA, USA).

    Techniques: Cell Culture, Incubation, Labeling, Expressing, Flow Cytometry, Cytometry

    The influence of TGF-β1 on the regulation of CD4 + T cell activation following curcumin treatment. CD4 + T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (Act.; 1∶10 for bead-to-cell ratio) or with 2 µg/mL of curcumin (Cur.) for 3 days. (A) Cell culture supernatants were collected at the indicated time point and total levels of active TGF-β were determined using an ELISA. (B) After 3 days of culture, cells were collected, washed with PBS and then transferred to a new cell culture plate in order to re-culture the cells in fresh media with or without a TGF-βRI kinase inhibitor (5 µg/mL) for an additional 3 days. The percentage of CD25 + , IFN-γ + , IL-10 + , IL-13 + and IL-17 + cells was determined by flow cytometric analysis. For detection of cytokine-producing cells, the cells were re-stimulated with PMA and ionomycin plus Brefeldin A for 5 hours. Data are presented as the mean ± SD. * P

    Journal: PLoS ONE

    Article Title: Curcumin Inhibits CD4+ T Cell Activation, but Augments CD69 Expression and TGF-?1-Mediated Generation of Regulatory T Cells at Late Phase

    doi: 10.1371/journal.pone.0062300

    Figure Lengend Snippet: The influence of TGF-β1 on the regulation of CD4 + T cell activation following curcumin treatment. CD4 + T cells were cultured in the presence of anti-CD2/CD3/CD28 antibody-coated beads only (Act.; 1∶10 for bead-to-cell ratio) or with 2 µg/mL of curcumin (Cur.) for 3 days. (A) Cell culture supernatants were collected at the indicated time point and total levels of active TGF-β were determined using an ELISA. (B) After 3 days of culture, cells were collected, washed with PBS and then transferred to a new cell culture plate in order to re-culture the cells in fresh media with or without a TGF-βRI kinase inhibitor (5 µg/mL) for an additional 3 days. The percentage of CD25 + , IFN-γ + , IL-10 + , IL-13 + and IL-17 + cells was determined by flow cytometric analysis. For detection of cytokine-producing cells, the cells were re-stimulated with PMA and ionomycin plus Brefeldin A for 5 hours. Data are presented as the mean ± SD. * P

    Article Snippet: U0126 (ERK inhibitor), SP600125 (JNK inhibitor), SB203580 (p38 MAPK inhibitor) and a TGF-β receptor I (TGF-βRI) kinase inhibitor were obtained from Calbiochem (San Diego, CA, USA).

    Techniques: Activation Assay, Cell Culture, Activated Clotting Time Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

    NUAK1 and NUAK2 are transcriptionally induced by TGFβ. A , real-time qRT-PCR analysis of NUAK1 mRNA normalized to HPRT1 mRNA from AG1523 cells after treatment with cycloheximide (20 μ m ) or an equivalent volume of PBS as negative control for 1 h followed by TGFβ (1 ng/ml) stimulation for 5 h. On the right , corresponding immunoblot for NUAK1, phospho-SMAD2 and total SMAD2 under the conditions used in the samples for real-time qRT-PCR. Molecular size markers in kDa are shown. One representative experiment of two is shown. B , real-time qRT-PCR analysis of Nuak2 mRNA normalized to Gapdh mRNA from NMuMG cells after treatment with vehicle or cycloheximide for 15 min followed by TGFβ (5 ng/ml) stimulation for 1 h. Immunoblots of NUAK2, phospho-SMAD2, and total SMAD2 proteins serve as controls for the RNA analysis. SMAD2 serves as a protein-loading control. Molecular size markers in kDa are shown. C , NMuMG cells were pretreated with vehicle or actinomycin D for 1 h before treatment with TGFβ (5 ng/ml) for 1 h. Nuak2 mRNA was normalized to Gapdh mRNA as measured by real-time qRT-PCR. D , immunoblotting of NUAK1 and SMAD4. AG1523 cells were stimulated with TGFβ (1 ng/ml) for 5 h. Ponceau-S staining of the immunoblot serves as a protein-loading control. Molecular size markers in kDa are shown. E , mRNA expression of Nuak2 , Gadd45 γ, and Smad4 in NMuMG cells after treatment with control or Smad4 siRNA and TGFβ (5 ng/ml) stimulation for 1 h. F , immunoblots of NUAK2, PAI-1, SMAD4, and β-actin proteins in MDA-MB-468 cells transiently infected with Adex-LacZ or Adex-SMAD4 (the latter at two different multiplicities of infection, 1 and 4) prior to cell starvation and stimulation with TGFβ (5 ng/ml) for 24 h. β-Actin serves as protein-loading control; a star shows nonspecific protein bands. Molecular size markers in kDa are shown. G , immunoblotting of NUAK1 and NUAK2 in AG1523 cells after treatment with TGFβ (5 ng/ml) for 3 h in the presence of TβRI kinase inhibitors LY2157299 (5 μ m ) or SB505124 (2.5 μ m ) or DMSO (0.1%). Molecular size markers in kDa are shown. H , immunoblots of NUAK2, phospho-ERK1/2, phospho-SMAD2, phospho-SMAD3, SMAD4, and β-actin proteins in NMuMG cells serum-starved overnight, followed by pretreatment with the indicated inhibitors or vehicle (DMSO) for 1 h prior to stimulation with TGFβ (1 ng/ml) for 6 h. β-Actin serves as protein-loading control. Molecular size markers in kDa are shown. Data are presented as mean ± S.E. ( error bars ) after performing at least three independent experiments. Asterisks imply significant differences compared with controls: *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Transforming growth factor β (TGFβ) induces NUAK kinase expression to fine-tune its signaling output

    doi: 10.1074/jbc.RA118.004984

    Figure Lengend Snippet: NUAK1 and NUAK2 are transcriptionally induced by TGFβ. A , real-time qRT-PCR analysis of NUAK1 mRNA normalized to HPRT1 mRNA from AG1523 cells after treatment with cycloheximide (20 μ m ) or an equivalent volume of PBS as negative control for 1 h followed by TGFβ (1 ng/ml) stimulation for 5 h. On the right , corresponding immunoblot for NUAK1, phospho-SMAD2 and total SMAD2 under the conditions used in the samples for real-time qRT-PCR. Molecular size markers in kDa are shown. One representative experiment of two is shown. B , real-time qRT-PCR analysis of Nuak2 mRNA normalized to Gapdh mRNA from NMuMG cells after treatment with vehicle or cycloheximide for 15 min followed by TGFβ (5 ng/ml) stimulation for 1 h. Immunoblots of NUAK2, phospho-SMAD2, and total SMAD2 proteins serve as controls for the RNA analysis. SMAD2 serves as a protein-loading control. Molecular size markers in kDa are shown. C , NMuMG cells were pretreated with vehicle or actinomycin D for 1 h before treatment with TGFβ (5 ng/ml) for 1 h. Nuak2 mRNA was normalized to Gapdh mRNA as measured by real-time qRT-PCR. D , immunoblotting of NUAK1 and SMAD4. AG1523 cells were stimulated with TGFβ (1 ng/ml) for 5 h. Ponceau-S staining of the immunoblot serves as a protein-loading control. Molecular size markers in kDa are shown. E , mRNA expression of Nuak2 , Gadd45 γ, and Smad4 in NMuMG cells after treatment with control or Smad4 siRNA and TGFβ (5 ng/ml) stimulation for 1 h. F , immunoblots of NUAK2, PAI-1, SMAD4, and β-actin proteins in MDA-MB-468 cells transiently infected with Adex-LacZ or Adex-SMAD4 (the latter at two different multiplicities of infection, 1 and 4) prior to cell starvation and stimulation with TGFβ (5 ng/ml) for 24 h. β-Actin serves as protein-loading control; a star shows nonspecific protein bands. Molecular size markers in kDa are shown. G , immunoblotting of NUAK1 and NUAK2 in AG1523 cells after treatment with TGFβ (5 ng/ml) for 3 h in the presence of TβRI kinase inhibitors LY2157299 (5 μ m ) or SB505124 (2.5 μ m ) or DMSO (0.1%). Molecular size markers in kDa are shown. H , immunoblots of NUAK2, phospho-ERK1/2, phospho-SMAD2, phospho-SMAD3, SMAD4, and β-actin proteins in NMuMG cells serum-starved overnight, followed by pretreatment with the indicated inhibitors or vehicle (DMSO) for 1 h prior to stimulation with TGFβ (1 ng/ml) for 6 h. β-Actin serves as protein-loading control. Molecular size markers in kDa are shown. Data are presented as mean ± S.E. ( error bars ) after performing at least three independent experiments. Asterisks imply significant differences compared with controls: *, p

    Article Snippet: TβRI kinase inhibitor GW6604 was used at a final concentration of 3 μm and was synthesized by the Ludwig Cancer Research Ltd. TβRI kinase inhibitor LY2157299 (Cayman Chemical Co., Stockholm, Sweden) was used at a final concentration of 5 μm , whereas TβRI kinase inhibitor SB505124 (Sigma-Aldrich Sweden AB) was applied at a concentration of 2.5 μm .

    Techniques: Quantitative RT-PCR, Negative Control, Western Blot, Staining, Expressing, Multiple Displacement Amplification, Infection

    NUAK1 and NUAK2 are transcriptionally induced by TGFβ. A , real-time qRT-PCR analysis of NUAK1 mRNA normalized to HPRT1 mRNA from AG1523 cells after treatment with cycloheximide (20 μ m ) or an equivalent volume of PBS as negative control for 1 h followed by TGFβ (1 ng/ml) stimulation for 5 h. On the right , corresponding immunoblot for NUAK1, phospho-SMAD2 and total SMAD2 under the conditions used in the samples for real-time qRT-PCR. Molecular size markers in kDa are shown. One representative experiment of two is shown. B , real-time qRT-PCR analysis of Nuak2 mRNA normalized to Gapdh mRNA from NMuMG cells after treatment with vehicle or cycloheximide for 15 min followed by TGFβ (5 ng/ml) stimulation for 1 h. Immunoblots of NUAK2, phospho-SMAD2, and total SMAD2 proteins serve as controls for the RNA analysis. SMAD2 serves as a protein-loading control. Molecular size markers in kDa are shown. C , NMuMG cells were pretreated with vehicle or actinomycin D for 1 h before treatment with TGFβ (5 ng/ml) for 1 h. Nuak2 mRNA was normalized to Gapdh mRNA as measured by real-time qRT-PCR. D , immunoblotting of NUAK1 and SMAD4. AG1523 cells were stimulated with TGFβ (1 ng/ml) for 5 h. Ponceau-S staining of the immunoblot serves as a protein-loading control. Molecular size markers in kDa are shown. E , mRNA expression of Nuak2 , Gadd45 γ, and Smad4 in NMuMG cells after treatment with control or Smad4 siRNA and TGFβ (5 ng/ml) stimulation for 1 h. F , immunoblots of NUAK2, PAI-1, SMAD4, and β-actin proteins in MDA-MB-468 cells transiently infected with Adex-LacZ or Adex-SMAD4 (the latter at two different multiplicities of infection, 1 and 4) prior to cell starvation and stimulation with TGFβ (5 ng/ml) for 24 h. β-Actin serves as protein-loading control; a star shows nonspecific protein bands. Molecular size markers in kDa are shown. G , immunoblotting of NUAK1 and NUAK2 in AG1523 cells after treatment with TGFβ (5 ng/ml) for 3 h in the presence of TβRI kinase inhibitors LY2157299 (5 μ m ) or SB505124 (2.5 μ m ) or DMSO (0.1%). Molecular size markers in kDa are shown. H , immunoblots of NUAK2, phospho-ERK1/2, phospho-SMAD2, phospho-SMAD3, SMAD4, and β-actin proteins in NMuMG cells serum-starved overnight, followed by pretreatment with the indicated inhibitors or vehicle (DMSO) for 1 h prior to stimulation with TGFβ (1 ng/ml) for 6 h. β-Actin serves as protein-loading control. Molecular size markers in kDa are shown. Data are presented as mean ± S.E. ( error bars ) after performing at least three independent experiments. Asterisks imply significant differences compared with controls: *, p

    Journal: The Journal of Biological Chemistry

    Article Title: Transforming growth factor β (TGFβ) induces NUAK kinase expression to fine-tune its signaling output

    doi: 10.1074/jbc.RA118.004984

    Figure Lengend Snippet: NUAK1 and NUAK2 are transcriptionally induced by TGFβ. A , real-time qRT-PCR analysis of NUAK1 mRNA normalized to HPRT1 mRNA from AG1523 cells after treatment with cycloheximide (20 μ m ) or an equivalent volume of PBS as negative control for 1 h followed by TGFβ (1 ng/ml) stimulation for 5 h. On the right , corresponding immunoblot for NUAK1, phospho-SMAD2 and total SMAD2 under the conditions used in the samples for real-time qRT-PCR. Molecular size markers in kDa are shown. One representative experiment of two is shown. B , real-time qRT-PCR analysis of Nuak2 mRNA normalized to Gapdh mRNA from NMuMG cells after treatment with vehicle or cycloheximide for 15 min followed by TGFβ (5 ng/ml) stimulation for 1 h. Immunoblots of NUAK2, phospho-SMAD2, and total SMAD2 proteins serve as controls for the RNA analysis. SMAD2 serves as a protein-loading control. Molecular size markers in kDa are shown. C , NMuMG cells were pretreated with vehicle or actinomycin D for 1 h before treatment with TGFβ (5 ng/ml) for 1 h. Nuak2 mRNA was normalized to Gapdh mRNA as measured by real-time qRT-PCR. D , immunoblotting of NUAK1 and SMAD4. AG1523 cells were stimulated with TGFβ (1 ng/ml) for 5 h. Ponceau-S staining of the immunoblot serves as a protein-loading control. Molecular size markers in kDa are shown. E , mRNA expression of Nuak2 , Gadd45 γ, and Smad4 in NMuMG cells after treatment with control or Smad4 siRNA and TGFβ (5 ng/ml) stimulation for 1 h. F , immunoblots of NUAK2, PAI-1, SMAD4, and β-actin proteins in MDA-MB-468 cells transiently infected with Adex-LacZ or Adex-SMAD4 (the latter at two different multiplicities of infection, 1 and 4) prior to cell starvation and stimulation with TGFβ (5 ng/ml) for 24 h. β-Actin serves as protein-loading control; a star shows nonspecific protein bands. Molecular size markers in kDa are shown. G , immunoblotting of NUAK1 and NUAK2 in AG1523 cells after treatment with TGFβ (5 ng/ml) for 3 h in the presence of TβRI kinase inhibitors LY2157299 (5 μ m ) or SB505124 (2.5 μ m ) or DMSO (0.1%). Molecular size markers in kDa are shown. H , immunoblots of NUAK2, phospho-ERK1/2, phospho-SMAD2, phospho-SMAD3, SMAD4, and β-actin proteins in NMuMG cells serum-starved overnight, followed by pretreatment with the indicated inhibitors or vehicle (DMSO) for 1 h prior to stimulation with TGFβ (1 ng/ml) for 6 h. β-Actin serves as protein-loading control. Molecular size markers in kDa are shown. Data are presented as mean ± S.E. ( error bars ) after performing at least three independent experiments. Asterisks imply significant differences compared with controls: *, p

    Article Snippet: TβRI kinase inhibitor GW6604 was used at a final concentration of 3 μm and was synthesized by the Ludwig Cancer Research Ltd. TβRI kinase inhibitor LY2157299 (Cayman Chemical Co., Stockholm, Sweden) was used at a final concentration of 5 μm , whereas TβRI kinase inhibitor SB505124 (Sigma-Aldrich Sweden AB) was applied at a concentration of 2.5 μm .

    Techniques: Quantitative RT-PCR, Negative Control, Western Blot, Staining, Expressing, Multiple Displacement Amplification, Infection

    GPR50 inhibits cell proliferation and tumor growth in MDA-MB-231 cells. a Expression of GPR50Δ4 and GPR50wt in lysates of MDA-MB-231 cell pools revealed by western blot. b An equal number of MDA-MB-231 cells expressing Mock, GPR50Δ4 or GPR50wt were seeded into 96-well plates, starved and stimulated with TGFβ (2 ng/mL) and transfected with either siRNA against control (si-Ctrl) or TβRI (si-TβRI). The proliferation rate over total amount of cells was measured with the MTT assay 5 days after starvation (Mean ± s.e.m., n = 5 independent experiments, * p

    Journal: Nature Communications

    Article Title: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development

    doi: 10.1038/s41467-018-03609-x

    Figure Lengend Snippet: GPR50 inhibits cell proliferation and tumor growth in MDA-MB-231 cells. a Expression of GPR50Δ4 and GPR50wt in lysates of MDA-MB-231 cell pools revealed by western blot. b An equal number of MDA-MB-231 cells expressing Mock, GPR50Δ4 or GPR50wt were seeded into 96-well plates, starved and stimulated with TGFβ (2 ng/mL) and transfected with either siRNA against control (si-Ctrl) or TβRI (si-TβRI). The proliferation rate over total amount of cells was measured with the MTT assay 5 days after starvation (Mean ± s.e.m., n = 5 independent experiments, * p

    Article Snippet: Precipitates were incubated with a TβRI Kinase (TGFβR1 Kinase Enzyme System, Promega) and kinase activity was measured by incubating with the delivered kinase substrate (Smad3 Peptide) and the use of the ADP-Glo™ Kinase Assay kit (Promega) according to the manufacturer’s instructions.

    Techniques: Multiple Displacement Amplification, Expressing, Western Blot, Transfection, MTT Assay

    Proposed Model of GPR50 action on TβRI-mediated signaling In the basal state (upper part), TβRI and TβRII, each form homodimers, that are apart from each other. TβRI is stabilized in its inhibitory confirmation by FKBP12. The R-Smads are non-phosphorylated in the cytosol and no transcription of target genes occurs. In the classical activation mode (lower left side), TGFβ binds to TβRII, which enables recruitment of TβRI into the complex. TβRII phosphorylates TβRI in the GS domain, FKBP12 dissociates from the complex and R-Smad2/3 becomes phosphorylated by TβRI after recruitment into the complex. Phosphorylated R-Smad2/3 dissociates and forms a complex with Smad4, which translocates into the nucleus, and regulates target gene expression. In the case that TβRI forms a complex with GPR50 (and not with TβRII) (lower right side), GPR50 induces the dissociation of FKBP12 from TβRI due to a similar motif of amino acids H87 and P88 in its C-tail on positions 498 and 499. The GPR50/TβRI complex then constitutively activates the classical and non-canonical Smad signaling pathways

    Journal: Nature Communications

    Article Title: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development

    doi: 10.1038/s41467-018-03609-x

    Figure Lengend Snippet: Proposed Model of GPR50 action on TβRI-mediated signaling In the basal state (upper part), TβRI and TβRII, each form homodimers, that are apart from each other. TβRI is stabilized in its inhibitory confirmation by FKBP12. The R-Smads are non-phosphorylated in the cytosol and no transcription of target genes occurs. In the classical activation mode (lower left side), TGFβ binds to TβRII, which enables recruitment of TβRI into the complex. TβRII phosphorylates TβRI in the GS domain, FKBP12 dissociates from the complex and R-Smad2/3 becomes phosphorylated by TβRI after recruitment into the complex. Phosphorylated R-Smad2/3 dissociates and forms a complex with Smad4, which translocates into the nucleus, and regulates target gene expression. In the case that TβRI forms a complex with GPR50 (and not with TβRII) (lower right side), GPR50 induces the dissociation of FKBP12 from TβRI due to a similar motif of amino acids H87 and P88 in its C-tail on positions 498 and 499. The GPR50/TβRI complex then constitutively activates the classical and non-canonical Smad signaling pathways

    Article Snippet: Precipitates were incubated with a TβRI Kinase (TGFβR1 Kinase Enzyme System, Promega) and kinase activity was measured by incubating with the delivered kinase substrate (Smad3 Peptide) and the use of the ADP-Glo™ Kinase Assay kit (Promega) according to the manufacturer’s instructions.

    Techniques: Activation Assay, Expressing

    GPR50 interacts with TβRI and its expression is upregulated by TGFβ. a Tandem affinity purification of naive HEK293T cells stably expressing GPR50Δ4-TAP. After purification, mass spectrometry was employed for protein identification. b . c Confocal images of GPR50 (red) and TβRI (green) staining in primary rat tanycytes (scale: 10 µm). d Colocalization of GPR50 (red) and TβRI (green) in NCI-H520 cells. (scale: 10 µm). e Co-immunoprecipitation of GRP50 and TβRI in lysates of primary rat tanycyte cultures. Lysates with IgG served as negative control. f Co-immunoprecipitation of GRP50 and TβRI in the lysates of NCI-H520 after silencing either GPR50 (si-GPR50) or TβRI (si-TβRI). Control si-RNA (si-Ctrl) served as control. g , h Co-immunoprecipitation of GRP50 and TβRI in lysates of MDA-MB231 cells ( g ) and cortex ( h ) isolated from wild type (wt) or GPR50ko mice. IgG served as negative control. i Upper part depicts schematic representation of BRET assay to study the interaction between TβRI-Rluc8 and GPR50-YFP or TβRI-YFP (left and middle scheme) and right scheme between TβRI-Rluc8 and TβRII-YFP. Lower part shows BRET donor saturation curves in HEK293T cells (left: constant expression level of TβRI-Rluc8 and increasing levels of TβRI-YFP, GPR50Δ4-YFP or GPR50wt-YFP; right: constant expression level of TβRI-Rluc8 and increasing levels of GPR50Δ4-YFP or TβRII-YFP with TGFβ stimulation (0.6 nM, 30 min at 37 °C)). IR-YFP and OBRa-YFP served as negative control. BRET signals were normalized to BRET max values. Curves are obtained from three independent experiments performed in triplicates. j NCI-H520 cells were starved and stimulated for 24 h with TGFβ (2 ng/mL). GPR50 expression was checked by Immunoblotting and Q-PCR. (Mean ± s.e.m., n = 3 independent experiments, * p

    Journal: Nature Communications

    Article Title: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development

    doi: 10.1038/s41467-018-03609-x

    Figure Lengend Snippet: GPR50 interacts with TβRI and its expression is upregulated by TGFβ. a Tandem affinity purification of naive HEK293T cells stably expressing GPR50Δ4-TAP. After purification, mass spectrometry was employed for protein identification. b . c Confocal images of GPR50 (red) and TβRI (green) staining in primary rat tanycytes (scale: 10 µm). d Colocalization of GPR50 (red) and TβRI (green) in NCI-H520 cells. (scale: 10 µm). e Co-immunoprecipitation of GRP50 and TβRI in lysates of primary rat tanycyte cultures. Lysates with IgG served as negative control. f Co-immunoprecipitation of GRP50 and TβRI in the lysates of NCI-H520 after silencing either GPR50 (si-GPR50) or TβRI (si-TβRI). Control si-RNA (si-Ctrl) served as control. g , h Co-immunoprecipitation of GRP50 and TβRI in lysates of MDA-MB231 cells ( g ) and cortex ( h ) isolated from wild type (wt) or GPR50ko mice. IgG served as negative control. i Upper part depicts schematic representation of BRET assay to study the interaction between TβRI-Rluc8 and GPR50-YFP or TβRI-YFP (left and middle scheme) and right scheme between TβRI-Rluc8 and TβRII-YFP. Lower part shows BRET donor saturation curves in HEK293T cells (left: constant expression level of TβRI-Rluc8 and increasing levels of TβRI-YFP, GPR50Δ4-YFP or GPR50wt-YFP; right: constant expression level of TβRI-Rluc8 and increasing levels of GPR50Δ4-YFP or TβRII-YFP with TGFβ stimulation (0.6 nM, 30 min at 37 °C)). IR-YFP and OBRa-YFP served as negative control. BRET signals were normalized to BRET max values. Curves are obtained from three independent experiments performed in triplicates. j NCI-H520 cells were starved and stimulated for 24 h with TGFβ (2 ng/mL). GPR50 expression was checked by Immunoblotting and Q-PCR. (Mean ± s.e.m., n = 3 independent experiments, * p

    Article Snippet: Precipitates were incubated with a TβRI Kinase (TGFβR1 Kinase Enzyme System, Promega) and kinase activity was measured by incubating with the delivered kinase substrate (Smad3 Peptide) and the use of the ADP-Glo™ Kinase Assay kit (Promega) according to the manufacturer’s instructions.

    Techniques: Expressing, Affinity Purification, Stable Transfection, Purification, Mass Spectrometry, Staining, Immunoprecipitation, Negative Control, Multiple Displacement Amplification, Isolation, Mouse Assay, Bioluminescence Resonance Energy Transfer, Polymerase Chain Reaction

    GPR50 promotes ligand-independent activation of TβRI signaling. a HEK293T cells expressing myc-Smad3, and GPR50Δ4 or GPR50wt were starved overnight and stimulated with TGFβ (2 ng/mL; 1 h). Smad3 phosphorylation was checked. Similar results were obtained in at least two additional experiments. b p-Smad2 detection in NCI-H520 cells following the silencing of GPR50 and TβRI Control si-RNA (si-Ctrl) with and without TGFβ stimulation (2 ng/mL; 1 h) served as control. Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, *** p

    Journal: Nature Communications

    Article Title: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development

    doi: 10.1038/s41467-018-03609-x

    Figure Lengend Snippet: GPR50 promotes ligand-independent activation of TβRI signaling. a HEK293T cells expressing myc-Smad3, and GPR50Δ4 or GPR50wt were starved overnight and stimulated with TGFβ (2 ng/mL; 1 h). Smad3 phosphorylation was checked. Similar results were obtained in at least two additional experiments. b p-Smad2 detection in NCI-H520 cells following the silencing of GPR50 and TβRI Control si-RNA (si-Ctrl) with and without TGFβ stimulation (2 ng/mL; 1 h) served as control. Densitometric analysis of three independent experiments (Mean ± s.e.m., n = 3 independent experiments, *** p

    Article Snippet: Precipitates were incubated with a TβRI Kinase (TGFβR1 Kinase Enzyme System, Promega) and kinase activity was measured by incubating with the delivered kinase substrate (Smad3 Peptide) and the use of the ADP-Glo™ Kinase Assay kit (Promega) according to the manufacturer’s instructions.

    Techniques: Activation Assay, Expressing

    GPR50 phosphorylate and activate TβRI independently of TβRII. a SNU638 cells were transfected with the indicated plasmids, stimulated for 1 h with TGFβ (2 ng/mL) and pretreated or not overnight with SB431542 at 10 µM. p-Smad3 and total Smad3 levels and expression of transfected plasmids were determined by western blot in cell lysates. b Co-immunoprecipitation of GRP50 and TβRI in lysates of SNU638 cells expressing HA-TβRI alone or together with GPR50Δ4. Total lysates were used as expression control ( c ) SNU638 cells were transfected with the indicated plasmids, stimulated TGFβ (2 ng/mL, 1 h). Total TβRI was immunoprecipitated with anti-Flag antibody and immunoblotted for anti-TβRIp-S165. Below, the same blot was immunoblotted with anti-Flag to show the amount of TβRI precipitation in cell lysates. d Phospho-Smad3 and Smad2/3 levels were determined in lysates of SNU638 cells stimulated or not with TGFβ (2 ng/mL, 1 h) and expressing the indicated proteins (as verified by western blot). The myc-MT 2 melatonin receptor and the myc-MT 2

    Journal: Nature Communications

    Article Title: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development

    doi: 10.1038/s41467-018-03609-x

    Figure Lengend Snippet: GPR50 phosphorylate and activate TβRI independently of TβRII. a SNU638 cells were transfected with the indicated plasmids, stimulated for 1 h with TGFβ (2 ng/mL) and pretreated or not overnight with SB431542 at 10 µM. p-Smad3 and total Smad3 levels and expression of transfected plasmids were determined by western blot in cell lysates. b Co-immunoprecipitation of GRP50 and TβRI in lysates of SNU638 cells expressing HA-TβRI alone or together with GPR50Δ4. Total lysates were used as expression control ( c ) SNU638 cells were transfected with the indicated plasmids, stimulated TGFβ (2 ng/mL, 1 h). Total TβRI was immunoprecipitated with anti-Flag antibody and immunoblotted for anti-TβRIp-S165. Below, the same blot was immunoblotted with anti-Flag to show the amount of TβRI precipitation in cell lysates. d Phospho-Smad3 and Smad2/3 levels were determined in lysates of SNU638 cells stimulated or not with TGFβ (2 ng/mL, 1 h) and expressing the indicated proteins (as verified by western blot). The myc-MT 2 melatonin receptor and the myc-MT 2

    Article Snippet: Precipitates were incubated with a TβRI Kinase (TGFβR1 Kinase Enzyme System, Promega) and kinase activity was measured by incubating with the delivered kinase substrate (Smad3 Peptide) and the use of the ADP-Glo™ Kinase Assay kit (Promega) according to the manufacturer’s instructions.

    Techniques: Transfection, Expressing, Western Blot, Immunoprecipitation

    GPR50 competes with FKBP12 for the binding to TβRI. a (Left) HEK293T cells were transfected with Flag-TβRI alone or cotransfected with HA-TβRII (with TGFβ; 2 ng/mL; 1 h), myc-FKBP12 and either GPR50Δ4 or GPR50wt. Lysates were precipitated for FKBP12 using anti-myc antibody and blotted with an anti-Flag to reveal complex formation. Expression of myc-FKBP12, Flag-TβRI, HA-TβRII and GPR50 was determined in total lysates. (Right) Densitometric analysis of 3 independent experiments (Mean ± s.e.m., n = 3 independent experiments, ** p

    Journal: Nature Communications

    Article Title: The orphan GPR50 receptor promotes constitutive TGFβ receptor signaling and protects against cancer development

    doi: 10.1038/s41467-018-03609-x

    Figure Lengend Snippet: GPR50 competes with FKBP12 for the binding to TβRI. a (Left) HEK293T cells were transfected with Flag-TβRI alone or cotransfected with HA-TβRII (with TGFβ; 2 ng/mL; 1 h), myc-FKBP12 and either GPR50Δ4 or GPR50wt. Lysates were precipitated for FKBP12 using anti-myc antibody and blotted with an anti-Flag to reveal complex formation. Expression of myc-FKBP12, Flag-TβRI, HA-TβRII and GPR50 was determined in total lysates. (Right) Densitometric analysis of 3 independent experiments (Mean ± s.e.m., n = 3 independent experiments, ** p

    Article Snippet: Precipitates were incubated with a TβRI Kinase (TGFβR1 Kinase Enzyme System, Promega) and kinase activity was measured by incubating with the delivered kinase substrate (Smad3 Peptide) and the use of the ADP-Glo™ Kinase Assay kit (Promega) according to the manufacturer’s instructions.

    Techniques: Binding Assay, Transfection, Expressing

    Albumin-induced increase in matrix metalloproteinase (MMP)-9 occurs independently of the transforming growth factor (TGF)-β receptor. Representative zymogram and corresponding quantification of the level of MMP-9 release by astrocytes treated with BSA in the presence (+) or absence (−) of (a) the TGF-β receptor I inhibitor SB431542, or (b) the Smad pathway inhibitor SIS3; neither inhibition of the TGF-β receptor I inhibitor nor the Smad pathway inhibited albumin-induced increase in MMP-9. (c) Representative zymogram of the level of MMP-9 release by astrocytes treated with BSA and TGF-β1 (10 ng/mL). Data are representative of mean ± SEM of three independent experiments. *** P

    Journal: Journal of Neuroinflammation

    Article Title: Albumin induces upregulation of matrix metalloproteinase-9 in astrocytes via MAPK and reactive oxygen species-dependent pathways

    doi: 10.1186/1742-2094-9-68

    Figure Lengend Snippet: Albumin-induced increase in matrix metalloproteinase (MMP)-9 occurs independently of the transforming growth factor (TGF)-β receptor. Representative zymogram and corresponding quantification of the level of MMP-9 release by astrocytes treated with BSA in the presence (+) or absence (−) of (a) the TGF-β receptor I inhibitor SB431542, or (b) the Smad pathway inhibitor SIS3; neither inhibition of the TGF-β receptor I inhibitor nor the Smad pathway inhibited albumin-induced increase in MMP-9. (c) Representative zymogram of the level of MMP-9 release by astrocytes treated with BSA and TGF-β1 (10 ng/mL). Data are representative of mean ± SEM of three independent experiments. *** P

    Article Snippet: The role of the TGF-β receptor pathway was investigated by treating the cells with the TGF-β receptor I inhibitor SB431542 (Tocris, Ellisville, MO, USA) or the specific Smad3 inhibitor (SIS3, Calbiochem, San Diego, CA, USA).

    Techniques: Inhibition

    Albumin stimulates an increase in the release of tissue inhibitor of metalloproteinase (TIMP)-1 in the conditioned media by astrocytes. (a) Astrocytes exposed to BSA had a time-dependent increase in TIMP-1. (b) Levels of TIMP-1 in astrocytes treated with albumin in the presence (+) or absence (−) of the TGF-β receptor I inhibitor SB431542. (c) Levels in astrocytes treated with albumin in the presence of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (SB), the extracellular signal regulated protein kinase (ERK) pathway inhibitor PD98059 (PD), and the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (SP). (d) Levels in astrocytes treated with albumin in the presence (+) or absence (−) of SIS3, the Smad pathway inhibitor. (e) Levels of TIMP-1 in astrocytes treated with albumin in the presence (+) or absence (−) of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) or (f) the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). Data are representative of mean ± SEM of 3–4 independent experiments, and are expressed (B-F) as a percentage of the value of the BSA-treated group. ** P

    Journal: Journal of Neuroinflammation

    Article Title: Albumin induces upregulation of matrix metalloproteinase-9 in astrocytes via MAPK and reactive oxygen species-dependent pathways

    doi: 10.1186/1742-2094-9-68

    Figure Lengend Snippet: Albumin stimulates an increase in the release of tissue inhibitor of metalloproteinase (TIMP)-1 in the conditioned media by astrocytes. (a) Astrocytes exposed to BSA had a time-dependent increase in TIMP-1. (b) Levels of TIMP-1 in astrocytes treated with albumin in the presence (+) or absence (−) of the TGF-β receptor I inhibitor SB431542. (c) Levels in astrocytes treated with albumin in the presence of the p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 (SB), the extracellular signal regulated protein kinase (ERK) pathway inhibitor PD98059 (PD), and the c-Jun N-terminal kinase (JNK) inhibitor SP600125 (SP). (d) Levels in astrocytes treated with albumin in the presence (+) or absence (−) of SIS3, the Smad pathway inhibitor. (e) Levels of TIMP-1 in astrocytes treated with albumin in the presence (+) or absence (−) of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT) or (f) the NADPH oxidase inhibitor diphenyleneiodonium chloride (DPI). Data are representative of mean ± SEM of 3–4 independent experiments, and are expressed (B-F) as a percentage of the value of the BSA-treated group. ** P

    Article Snippet: The role of the TGF-β receptor pathway was investigated by treating the cells with the TGF-β receptor I inhibitor SB431542 (Tocris, Ellisville, MO, USA) or the specific Smad3 inhibitor (SIS3, Calbiochem, San Diego, CA, USA).

    Techniques:

    Increased expression of TβRI and TβRII in CD4 + CD25 – T cells of PARP-1 −/− mice. (A-C) Quantitative PCR analysis of the expression of Tgfbr1 and Tgfbr2 mRNA in (A) freshly isolated splenic CD4 + CD25 - T cells from PARP-1

    Journal: Blood

    Article Title: PARP-1 regulates expression of TGF-β receptors in T cells

    doi: 10.1182/blood-2013-05-503250

    Figure Lengend Snippet: Increased expression of TβRI and TβRII in CD4 + CD25 – T cells of PARP-1 −/− mice. (A-C) Quantitative PCR analysis of the expression of Tgfbr1 and Tgfbr2 mRNA in (A) freshly isolated splenic CD4 + CD25 - T cells from PARP-1

    Article Snippet: TβRI kinase inhibitor II was from Calbiochem.

    Techniques: Expressing, Mouse Assay, Real-time Polymerase Chain Reaction, Isolation

    GDF-15 effects on the canonical TGF-β signaling pathway in glioma cells A. LNT-229 or LN-308 cells were exposed to GDF-15 (100 ng/ml), BMP-4 (5 ng/ml) or TGF-β 2 (10 ng/ml) as indicated for 24 h, harvested and assessed for pSmad2, Smad2/3, pSmad1/5/8 and Smad5 levels by immunoblot. B. LNT-229 or LN-308 cells transfected with the pGL3-SBE4-Luc reporter plasmid were exposed to TGF-β 2 , GDF-15, SD-208 or combinations thereof as indicated for 24 h, and analyzed for firefly/renilla luciferase activity. C. LNT-229 and LN-308 cells transfected with the pGL3-SBE4-Luc reporter plasmid were pre-exposed to GDF-15 for 1.5 h or not as indicated, subsequently treated with TGF-β 2 for 24 h and analyzed as in (B) (* p

    Journal: Oncotarget

    Article Title: Control of glioma cell migration and invasiveness by GDF-15

    doi: 10.18632/oncotarget.6816

    Figure Lengend Snippet: GDF-15 effects on the canonical TGF-β signaling pathway in glioma cells A. LNT-229 or LN-308 cells were exposed to GDF-15 (100 ng/ml), BMP-4 (5 ng/ml) or TGF-β 2 (10 ng/ml) as indicated for 24 h, harvested and assessed for pSmad2, Smad2/3, pSmad1/5/8 and Smad5 levels by immunoblot. B. LNT-229 or LN-308 cells transfected with the pGL3-SBE4-Luc reporter plasmid were exposed to TGF-β 2 , GDF-15, SD-208 or combinations thereof as indicated for 24 h, and analyzed for firefly/renilla luciferase activity. C. LNT-229 and LN-308 cells transfected with the pGL3-SBE4-Luc reporter plasmid were pre-exposed to GDF-15 for 1.5 h or not as indicated, subsequently treated with TGF-β 2 for 24 h and analyzed as in (B) (* p

    Article Snippet: The TGF-β receptor I kinase inhibitor SD-208 was kindly provided by Scios Inc (Fremont, CA).

    Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay

    miR-27a directly targets TGF-βRI and represses the TGF-β signaling. a qRT-PCR analysis of TGF-βRI and TGF-βRII expression in cervical cancer cell lines versus normal cervical epithelia. b qRT-PCR. TGF-βRI expression was downregulated by miR-27a agomir in HeLa, but not in SiHa. c qRT-PCR. TGF-βRII levels in three cervical cancer cell lines were not affected by agomir transfection. d Results of double-luciferase reporter assays show the luciferase activity of the wild type TGF-βRI 3′-UTR (TGF-βRI-WT) and the mutant one (TGF-βRI-Mut) in the absence or presence of hsp-miR-27a-3p mimics. e – h qRT-PCR. Expression levels of TGF-βRI, SMAD2, SMAD3, and SMAD4 in cervical cancer cells that were transfected with agomir NC or miR-27a agomir. i Western blot. TGF-βRI, SMAD3, and p-SMAD3 were downregulated by miR-27a agomir transfection. ** P

    Journal: Cell Death & Disease

    Article Title: miR-27a inhibits cervical adenocarcinoma progression by downregulating the TGF-βRI signaling pathway

    doi: 10.1038/s41419-018-0431-2

    Figure Lengend Snippet: miR-27a directly targets TGF-βRI and represses the TGF-β signaling. a qRT-PCR analysis of TGF-βRI and TGF-βRII expression in cervical cancer cell lines versus normal cervical epithelia. b qRT-PCR. TGF-βRI expression was downregulated by miR-27a agomir in HeLa, but not in SiHa. c qRT-PCR. TGF-βRII levels in three cervical cancer cell lines were not affected by agomir transfection. d Results of double-luciferase reporter assays show the luciferase activity of the wild type TGF-βRI 3′-UTR (TGF-βRI-WT) and the mutant one (TGF-βRI-Mut) in the absence or presence of hsp-miR-27a-3p mimics. e – h qRT-PCR. Expression levels of TGF-βRI, SMAD2, SMAD3, and SMAD4 in cervical cancer cells that were transfected with agomir NC or miR-27a agomir. i Western blot. TGF-βRI, SMAD3, and p-SMAD3 were downregulated by miR-27a agomir transfection. ** P

    Article Snippet: The TGF-βRI kinase activity inhibitor A83-01 was purchased from Tocris Bioscience (Bristol, UK), and the cells were treated with a final concentration of 10 μM for 72 h .

    Techniques: Quantitative RT-PCR, Expressing, Transfection, Luciferase, Activity Assay, Mutagenesis, Western Blot

    miR-27a agomir inhibits the growth of cervical cancer xenografts in vivo. Mice harboring subcutaneous tumors derived from HeLa cells received intratumoral injection of PBS, agomir NC, or miR-27a agomir ( N = 7/group). a The tumor size in the agomir group is significantly smaller than the NC group and the PBS group (23 days after inoculation). b The curves of relative tumor volume show a significantly slower tumor growth in the agomir group compared with controls. c HE staining and immunohistochemical detection of TGF-βRI, SMAD2, SMAD3, p-SMAD3, and Ki-67 in the xenografts (bar, 200 µm). d – h Quantification of the immunohistochemistry results. *** P

    Journal: Cell Death & Disease

    Article Title: miR-27a inhibits cervical adenocarcinoma progression by downregulating the TGF-βRI signaling pathway

    doi: 10.1038/s41419-018-0431-2

    Figure Lengend Snippet: miR-27a agomir inhibits the growth of cervical cancer xenografts in vivo. Mice harboring subcutaneous tumors derived from HeLa cells received intratumoral injection of PBS, agomir NC, or miR-27a agomir ( N = 7/group). a The tumor size in the agomir group is significantly smaller than the NC group and the PBS group (23 days after inoculation). b The curves of relative tumor volume show a significantly slower tumor growth in the agomir group compared with controls. c HE staining and immunohistochemical detection of TGF-βRI, SMAD2, SMAD3, p-SMAD3, and Ki-67 in the xenografts (bar, 200 µm). d – h Quantification of the immunohistochemistry results. *** P

    Article Snippet: The TGF-βRI kinase activity inhibitor A83-01 was purchased from Tocris Bioscience (Bristol, UK), and the cells were treated with a final concentration of 10 μM for 72 h .

    Techniques: In Vivo, Mouse Assay, Derivative Assay, Injection, Staining, Immunohistochemistry

    miR-27a functions as a tumor suppressor through repressing TGF-βRI expression and TGF-β signaling. a – c qRT-PCR analysis of TGF-βRI, TGF-βRII, and SMAD3 expression in cervical cancer cells that were transfected with miR-27a agomir in combination with TGF-βRI expression vector or control vector. d Western blot analysis of TGF-βRI, TGF-βRII, SMAD3, and p-SMAD3 levels in cervical cancer cells that were transfected with miR-27a agomir in combination with TGF-βRI expression vector or control vector. A83-01, a TGF-β signaling inhibitor, serves as positive control. DMSO is solvent control of A83-01 treatment. e – i The co-transfection of miR-27a agomir and TGF-βRI expression vector relieves the tumor inhibition effects mediated by miR-27a agomir. e EdU assays. f CFSE proliferation assays. g Invasion assays. h Migration assays. i Flow cytometry apoptosis assays. *** P

    Journal: Cell Death & Disease

    Article Title: miR-27a inhibits cervical adenocarcinoma progression by downregulating the TGF-βRI signaling pathway

    doi: 10.1038/s41419-018-0431-2

    Figure Lengend Snippet: miR-27a functions as a tumor suppressor through repressing TGF-βRI expression and TGF-β signaling. a – c qRT-PCR analysis of TGF-βRI, TGF-βRII, and SMAD3 expression in cervical cancer cells that were transfected with miR-27a agomir in combination with TGF-βRI expression vector or control vector. d Western blot analysis of TGF-βRI, TGF-βRII, SMAD3, and p-SMAD3 levels in cervical cancer cells that were transfected with miR-27a agomir in combination with TGF-βRI expression vector or control vector. A83-01, a TGF-β signaling inhibitor, serves as positive control. DMSO is solvent control of A83-01 treatment. e – i The co-transfection of miR-27a agomir and TGF-βRI expression vector relieves the tumor inhibition effects mediated by miR-27a agomir. e EdU assays. f CFSE proliferation assays. g Invasion assays. h Migration assays. i Flow cytometry apoptosis assays. *** P

    Article Snippet: The TGF-βRI kinase activity inhibitor A83-01 was purchased from Tocris Bioscience (Bristol, UK), and the cells were treated with a final concentration of 10 μM for 72 h .

    Techniques: Expressing, Quantitative RT-PCR, Transfection, Plasmid Preparation, Western Blot, Positive Control, Cotransfection, Inhibition, Migration, Flow Cytometry, Cytometry

    miR-27a and TGF-βRI expression in human cervical cancer . miR-27a was detected by tissue microarray-ISH. TGF-βRI and p-SMAD3 were detected by tissue microarray-ICH. a Representative images of HE staining, ISH staining for miR-27a, and IHC staining for TGF-βRI and p-SMAD3 in cervical cancers (squamous cell carcinoma, N = 71; adenocarcinoma, N = 28) and normal cervix tissues ( N = 76). The region marked by a red rectangle is enlarged in the top-right corner of each image (400×). b Comparison of the miR-27a expression in cervical adenocarcinomas (CADCs) to that in normal cervix glandular epithelia. c Comparison of the miR-27a expression in cervical squamous cell carcinomas (CSCCs) to that in normal cervix squamous epithelia. d Comparison of the TGF-βRI expression in CADCs to that in normal cervix glandular epithelia. e Comparison of the TGF-βRI expression in CSCCs to that in normal cervix squamous epithelia. f Comparison of the p-SMAD3 level in CADCs to that in normal cervix glandular epithelia. g Comparison of the p-SMAD3 level in CSCCs to that in normal cervix squamous epithelia. h miR-27a expression is negatively correlated with TGF-βRI expression in CADC. i There is no significant correlation between miR-27a and TGF-βRI expression

    Journal: Cell Death & Disease

    Article Title: miR-27a inhibits cervical adenocarcinoma progression by downregulating the TGF-βRI signaling pathway

    doi: 10.1038/s41419-018-0431-2

    Figure Lengend Snippet: miR-27a and TGF-βRI expression in human cervical cancer . miR-27a was detected by tissue microarray-ISH. TGF-βRI and p-SMAD3 were detected by tissue microarray-ICH. a Representative images of HE staining, ISH staining for miR-27a, and IHC staining for TGF-βRI and p-SMAD3 in cervical cancers (squamous cell carcinoma, N = 71; adenocarcinoma, N = 28) and normal cervix tissues ( N = 76). The region marked by a red rectangle is enlarged in the top-right corner of each image (400×). b Comparison of the miR-27a expression in cervical adenocarcinomas (CADCs) to that in normal cervix glandular epithelia. c Comparison of the miR-27a expression in cervical squamous cell carcinomas (CSCCs) to that in normal cervix squamous epithelia. d Comparison of the TGF-βRI expression in CADCs to that in normal cervix glandular epithelia. e Comparison of the TGF-βRI expression in CSCCs to that in normal cervix squamous epithelia. f Comparison of the p-SMAD3 level in CADCs to that in normal cervix glandular epithelia. g Comparison of the p-SMAD3 level in CSCCs to that in normal cervix squamous epithelia. h miR-27a expression is negatively correlated with TGF-βRI expression in CADC. i There is no significant correlation between miR-27a and TGF-βRI expression

    Article Snippet: The TGF-βRI kinase activity inhibitor A83-01 was purchased from Tocris Bioscience (Bristol, UK), and the cells were treated with a final concentration of 10 μM for 72 h .

    Techniques: Expressing, Microarray, In Situ Hybridization, Staining, Immunohistochemistry

    TGF-β signaling is required for COL1 expression in human skin fibroblasts. a Fibroblasts were treated with vehicle ( Ctrl ) or TβRI inhibitor SB431542 (10 μM) and analyzed 48 h after treatment. COL1A1 mRNA ( left panel

    Journal: Age

    Article Title: Oxidative exposure impairs TGF-β pathway via reduction of type II receptor and SMAD3 in human skin fibroblasts

    doi: 10.1007/s11357-014-9623-6

    Figure Lengend Snippet: TGF-β signaling is required for COL1 expression in human skin fibroblasts. a Fibroblasts were treated with vehicle ( Ctrl ) or TβRI inhibitor SB431542 (10 μM) and analyzed 48 h after treatment. COL1A1 mRNA ( left panel

    Article Snippet: For some studies, cultured dermal fibroblasts were treated with either NAC (10 mM, Sigma, St. Louis, MO) alone or NAC together with TβRI kinase inhibitor SB431542 (10 μM; Tocris Bioscience, Ellisville, MO) every other day, starting 1 or 7 days after oxidative exposure.

    Techniques: Expressing

    Induction of integrin αvβ6 expression requires TGF-β signaling and viral DNA replication A: Infected HUVECs were cultured with or without chicken anti-TGF-β polyclonal antibody (20 μg/ml), chicken IgY isotype control antibody (control Ab, 20 μg/ml), the ALK5 kinase inhibitor SB431542 (0.5 μmol/L), or the vehicle DMSO for 7 days, and surface expression of integrin αvβ6 was analyzed by flow cytometric analysis. Typical histograms are shown. Shaded areas represent expression of specific proteins. Lines represent isotype control. Experiments were repeated at least two times. B: Surface expression of integrin αvβ6 was analyzed by flow cytometric analysis at 7 days after infection with or without viral DNA polymerase inhibitors, Foscarnet, and phosphonoacetic acid (PAA). Typical histograms are shown. Shaded areas represent expression of specific proteins. Lines represent isotype control. Experiments were repeated six times. C: Active TGF-β was not produced by infected HUVECs in the presence of viral DNA polymerase inhibitors. Equal numbers of TMLC TGF-β reporter cells and control (cont.) or infected HUVECs were cultured for 16 to 24 hours at 7 days after infection. Relative luciferase activity in cell lysates was defined as the measured activity divided by TMLC baseline activity. Representative data (mean ± SE) are from four experiments done in triplicate.

    Journal: The American Journal of Pathology

    Article Title: Induction of an Epithelial Integrin αvβ6 in Human Cytomegalovirus-Infected Endothelial Cells Leads to Activation of Transforming Growth Factor-β1 and Increased Collagen Production

    doi: 10.2353/ajpath.2008.070448

    Figure Lengend Snippet: Induction of integrin αvβ6 expression requires TGF-β signaling and viral DNA replication A: Infected HUVECs were cultured with or without chicken anti-TGF-β polyclonal antibody (20 μg/ml), chicken IgY isotype control antibody (control Ab, 20 μg/ml), the ALK5 kinase inhibitor SB431542 (0.5 μmol/L), or the vehicle DMSO for 7 days, and surface expression of integrin αvβ6 was analyzed by flow cytometric analysis. Typical histograms are shown. Shaded areas represent expression of specific proteins. Lines represent isotype control. Experiments were repeated at least two times. B: Surface expression of integrin αvβ6 was analyzed by flow cytometric analysis at 7 days after infection with or without viral DNA polymerase inhibitors, Foscarnet, and phosphonoacetic acid (PAA). Typical histograms are shown. Shaded areas represent expression of specific proteins. Lines represent isotype control. Experiments were repeated six times. C: Active TGF-β was not produced by infected HUVECs in the presence of viral DNA polymerase inhibitors. Equal numbers of TMLC TGF-β reporter cells and control (cont.) or infected HUVECs were cultured for 16 to 24 hours at 7 days after infection. Relative luciferase activity in cell lysates was defined as the measured activity divided by TMLC baseline activity. Representative data (mean ± SE) are from four experiments done in triplicate.

    Article Snippet: TGF-β receptor I ALK5 kinase inhibitor SB431542 was purchased from Tocris Bioscience (Ellisville, MO).

    Techniques: Expressing, Infection, Cell Culture, Flow Cytometry, Produced, Luciferase, Activity Assay

    A: CMV-infected HUVECs induce Smad3 phosphorylation. Cell lysates from control (cont.) or infected (inf.) HUVECs at 3, 7, and 10 days after infection were fractionated by 10% SDS-PAGE and blotted on nitrocellulose. Phosphorylation of Smad3 (pSmad3) and Smad1/5 (pSmad1/5) was analyzed by immunoblotting using phospho-specific Smad3 and Smad1/5/8 (pSmad1/5) antibodies. Equal loading of the gels was confirmed using Smad2/3, Smad1, and Smad5 protein levels. B: Effects of anti-TGF-β antibody, anti-αvβ6 antibody (3G9), and ALK5 kinase inhibitor on Smad3 phosphorylation. Infected HUVECs were cultured without antibody (untreated) or with anti-TGF-β neutralizing antibody (1D11, 40 μg/ml), function-blocking anti-αvβ6 antibody (3G9, 80 μg/ml), mouse IgG1 isotype control antibody (control Ab, 80 μg/ml), the ALK5 kinase inhibitor SB431542 (2.5 μmol/L), or the vehicle DMSO for 8 days. Lysates were fractionated by 10% SDS-PAGE and blotted. Filters were incubated with antibodies to phosphorylated Smad3 (pSmad3), phosphorylated Smad1/5/8 (pSmad1/5), and Grb2 (loading control). Results are representative of at least four independent experiments.

    Journal: The American Journal of Pathology

    Article Title: Induction of an Epithelial Integrin αvβ6 in Human Cytomegalovirus-Infected Endothelial Cells Leads to Activation of Transforming Growth Factor-β1 and Increased Collagen Production

    doi: 10.2353/ajpath.2008.070448

    Figure Lengend Snippet: A: CMV-infected HUVECs induce Smad3 phosphorylation. Cell lysates from control (cont.) or infected (inf.) HUVECs at 3, 7, and 10 days after infection were fractionated by 10% SDS-PAGE and blotted on nitrocellulose. Phosphorylation of Smad3 (pSmad3) and Smad1/5 (pSmad1/5) was analyzed by immunoblotting using phospho-specific Smad3 and Smad1/5/8 (pSmad1/5) antibodies. Equal loading of the gels was confirmed using Smad2/3, Smad1, and Smad5 protein levels. B: Effects of anti-TGF-β antibody, anti-αvβ6 antibody (3G9), and ALK5 kinase inhibitor on Smad3 phosphorylation. Infected HUVECs were cultured without antibody (untreated) or with anti-TGF-β neutralizing antibody (1D11, 40 μg/ml), function-blocking anti-αvβ6 antibody (3G9, 80 μg/ml), mouse IgG1 isotype control antibody (control Ab, 80 μg/ml), the ALK5 kinase inhibitor SB431542 (2.5 μmol/L), or the vehicle DMSO for 8 days. Lysates were fractionated by 10% SDS-PAGE and blotted. Filters were incubated with antibodies to phosphorylated Smad3 (pSmad3), phosphorylated Smad1/5/8 (pSmad1/5), and Grb2 (loading control). Results are representative of at least four independent experiments.

    Article Snippet: TGF-β receptor I ALK5 kinase inhibitor SB431542 was purchased from Tocris Bioscience (Ellisville, MO).

    Techniques: Infection, SDS Page, Cell Culture, Blocking Assay, Incubation

    TGF-β signaling pathway. Bioactive TGF-β binds to the TGF-β type II receptor (TβRII), recruiting the TGF-β type I receptor (TβRI a.k.a. ALK5). TβRII phosphorylates and activates TβRI/ALK5,

    Journal: Cancer Microenvironment

    Article Title: TGF-? in the Bone Microenvironment: Role in Breast Cancer Metastases

    doi: 10.1007/s12307-011-0075-6

    Figure Lengend Snippet: TGF-β signaling pathway. Bioactive TGF-β binds to the TGF-β type II receptor (TβRII), recruiting the TGF-β type I receptor (TβRI a.k.a. ALK5). TβRII phosphorylates and activates TβRI/ALK5,

    Article Snippet: SD-208 and SD-093 (Scios Inc) are TβRI/ALK5 kinase inhibitors that were shown to inhibit migration, invasion and EMT in murine mammary epithelial (NMuMG) and carcinoma (4T1, R3T) cells [ ].

    Techniques:

    TGF-β signaling pathway. Bioactive TGF-β binds to the TGF-β type II receptor (TβRII), recruiting the TGF-β type I receptor (TβRI a.k.a. ALK5). TβRII phosphorylates and activates TβRI/ALK5,

    Journal: Cancer Microenvironment

    Article Title: TGF-? in the Bone Microenvironment: Role in Breast Cancer Metastases

    doi: 10.1007/s12307-011-0075-6

    Figure Lengend Snippet: TGF-β signaling pathway. Bioactive TGF-β binds to the TGF-β type II receptor (TβRII), recruiting the TGF-β type I receptor (TβRI a.k.a. ALK5). TβRII phosphorylates and activates TβRI/ALK5,

    Article Snippet: Ki26894, a TβRI/ALK5 kinase inhibitor developed by Kirin Brewery Company (Gunma, Japan), was shown to block TGF-β signaling, invasion and motility in MDA-MB-231-D bone tropic cells, including suppression of PTHrP and IL-11 [ ].

    Techniques:

    Generation of a novel nondiffusible TβRI kinase inhibitor. ( A ) Structure of pyrogallol (Pg), 3Abd, and derivatives. ( B ) A549 cells were stimulated with TGF-β1 for 30 minutes and lysates immunoblotted for p-Smad3, Smad3, and β-actin. Treatment without preincubation: 3Abd (0.5–10 μM); 2Abd or 4Abd (1, 10 μM). ( C ) A549 cells were stimulated with TGF-β1 for 48 hours and lysates immunoblotted for LOXL2, fibronectin, E-cadherin, Snail1, and β-actin. 3Abd was added at concentraions ranging from 0.5 to 10 μM. ( D ) Purified ALK5/TβRI catalytic domain kinase assay was performed with 10 doses of 3Abd or Pg starting from 100 μM. Kinase activity was indicated by 33 P-ATP signals, and IC 50 of 3Abd was calculated as approximately 3 μM. B – D are representative of 3 experiments with similar results. ( E ) SMAD-binding element (SBE) reporter–transfected NMuMG and A549 cells were seeded into a 96-well plate. Cocultured wells were seeded with 5,000 transfected NMuMG cells and 25,000 nontransfected A549 cells. Cells were pretreated with or without 1 μM corilagin, 1 μM EGCG, 10 μM 3Abd, or 5 μM SB431542 for 6 hours and stimulated with TGF-β1 overnight before lysis for luciferase assay. Data are presented as percent TGF-β1–induced SBE luciferase activity of DMSO control in log scale. Mean ± SD, n = 3. ( F ) Flag-tagged TβRI and TβRII were immunoprecipitated from A549 cells and in vitro kinase assay performed on beads exposed to lysate pretreated with corilagin or DMSO. The final reaction was eluted and analyzed by immunoblotting for phosphotyrosine and TβRI. The phosphotyrosine bands were quantified using ImageJ and normalized to DMSO control. Data represent mean ± SD. P value by unpaired 2-tailed t test of 6 separate experiments. ( G ) Schematic overview of mechanism. A trihydroxyphenolic-containing compound engages active LOXL2, initiating auto-oxidation of K731 and creating a key allysine inactivating the enzyme. In the process, a 3Abd-like metabolite is generated that then blocks TβRI kinase. The combined effects block pathological collagen accumulation.

    Journal: The Journal of Clinical Investigation

    Article Title: Fibroblast-specific inhibition of TGF- β1 signaling attenuates lung and tumor fibrosis

    doi: 10.1172/JCI94624

    Figure Lengend Snippet: Generation of a novel nondiffusible TβRI kinase inhibitor. ( A ) Structure of pyrogallol (Pg), 3Abd, and derivatives. ( B ) A549 cells were stimulated with TGF-β1 for 30 minutes and lysates immunoblotted for p-Smad3, Smad3, and β-actin. Treatment without preincubation: 3Abd (0.5–10 μM); 2Abd or 4Abd (1, 10 μM). ( C ) A549 cells were stimulated with TGF-β1 for 48 hours and lysates immunoblotted for LOXL2, fibronectin, E-cadherin, Snail1, and β-actin. 3Abd was added at concentraions ranging from 0.5 to 10 μM. ( D ) Purified ALK5/TβRI catalytic domain kinase assay was performed with 10 doses of 3Abd or Pg starting from 100 μM. Kinase activity was indicated by 33 P-ATP signals, and IC 50 of 3Abd was calculated as approximately 3 μM. B – D are representative of 3 experiments with similar results. ( E ) SMAD-binding element (SBE) reporter–transfected NMuMG and A549 cells were seeded into a 96-well plate. Cocultured wells were seeded with 5,000 transfected NMuMG cells and 25,000 nontransfected A549 cells. Cells were pretreated with or without 1 μM corilagin, 1 μM EGCG, 10 μM 3Abd, or 5 μM SB431542 for 6 hours and stimulated with TGF-β1 overnight before lysis for luciferase assay. Data are presented as percent TGF-β1–induced SBE luciferase activity of DMSO control in log scale. Mean ± SD, n = 3. ( F ) Flag-tagged TβRI and TβRII were immunoprecipitated from A549 cells and in vitro kinase assay performed on beads exposed to lysate pretreated with corilagin or DMSO. The final reaction was eluted and analyzed by immunoblotting for phosphotyrosine and TβRI. The phosphotyrosine bands were quantified using ImageJ and normalized to DMSO control. Data represent mean ± SD. P value by unpaired 2-tailed t test of 6 separate experiments. ( G ) Schematic overview of mechanism. A trihydroxyphenolic-containing compound engages active LOXL2, initiating auto-oxidation of K731 and creating a key allysine inactivating the enzyme. In the process, a 3Abd-like metabolite is generated that then blocks TβRI kinase. The combined effects block pathological collagen accumulation.

    Article Snippet: The authors thank Steve Chen from UCSF Small Molecule Discovery Center for assistance with high-throughput screening, Reaction Biology Corp. for performing the TβRI catalytic domain kinase assay, Eurofins Pharma Discovery Services UK Ltd. for performing protein and lipid kinase assays, David Morgan at Pliant Therapeutics for assistance with the kinase screen, and Julie Ren from Quintara Discovery for analyzing LC-MS/MS data.

    Techniques: Purification, Kinase Assay, Activity Assay, Binding Assay, Transfection, Lysis, Luciferase, Immunoprecipitation, In Vitro, Generated, Blocking Assay

    Galunisertib activities in 13 PDX samples and its correlations with TGF-βRI mRNA and pSMAD2 protein expression. ( a ) The in vivo efficacies were evaluated by ABC (see materials and methods ) values and compared to TGF-βRI mRNA expression (Affymetrix HGU133 Plus 2.0) and pSMAD2 protein expression (Western blotting; see Fig. 4 ) levels. ( b ) Correlation between TGF-βRI mRNA expression levels and ABC values (%). ( c ) Correlation between pSMAD2 protein expression levels and ABC values. P values ≤ 0.05 are considered significant

    Journal: Cellular Oncology (Dordrecht)

    Article Title: Anti-tumor activity of the TGF-β receptor kinase inhibitor galunisertib (LY2157299 monohydrate) in patient-derived tumor xenografts

    doi: 10.1007/s13402-014-0210-8

    Figure Lengend Snippet: Galunisertib activities in 13 PDX samples and its correlations with TGF-βRI mRNA and pSMAD2 protein expression. ( a ) The in vivo efficacies were evaluated by ABC (see materials and methods ) values and compared to TGF-βRI mRNA expression (Affymetrix HGU133 Plus 2.0) and pSMAD2 protein expression (Western blotting; see Fig. 4 ) levels. ( b ) Correlation between TGF-βRI mRNA expression levels and ABC values (%). ( c ) Correlation between pSMAD2 protein expression levels and ABC values. P values ≤ 0.05 are considered significant

    Article Snippet: Small molecule TGF-β receptor kinase inhibitor The small molecule LY2157299 monohydrate (galunisertib), targeting TGF-βRI serine/threonine kinase activity, was provided by Eli Lilly and Company, Indianapolis, USA.

    Techniques: Expressing, In Vivo, Western Blot

    Morphometric analyses of mechanisms and pathways in rAAV-transduced chondrocytes in situ . Explants were transduced with rAAV- lacZ or rAAV-hTGF-β as described in Figure 1 and maintained in culture for up to 90 days. The samples were then fixed and histologically processed at the denoted time points to monitor the % of cells immunostained for MMP-13 (A) , TIMP-1 (B) , TIMP-3 (C) , PTHrP (D) , β-catenin (E) , and the TGF-β receptor I (ALK1 and ALK5) (F and G , respectively ) . Data on the ALK1/ALK5 ratio are presented in (H) .

    Journal: Journal of Translational Medicine

    Article Title: rAAV-mediated overexpression of TGF-? stably restructures human osteoarthritic articular cartilage in situ

    doi: 10.1186/1479-5876-11-211

    Figure Lengend Snippet: Morphometric analyses of mechanisms and pathways in rAAV-transduced chondrocytes in situ . Explants were transduced with rAAV- lacZ or rAAV-hTGF-β as described in Figure 1 and maintained in culture for up to 90 days. The samples were then fixed and histologically processed at the denoted time points to monitor the % of cells immunostained for MMP-13 (A) , TIMP-1 (B) , TIMP-3 (C) , PTHrP (D) , β-catenin (E) , and the TGF-β receptor I (ALK1 and ALK5) (F and G , respectively ) . Data on the ALK1/ALK5 ratio are presented in (H) .

    Article Snippet: The anti-TGF-β (V), anti-MMP-13 (72B-01), anti-TIMP-1 (C-20) and -TIMP-3 (W-18), anti-parathyroid hormone-related protein (PTHrP) (1D1), anti-β-catenin (E-5), and anti-TGF-β receptor I (activin receptor-like kinase-1 ALK1: C-20; ALK5: T-19) antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany).

    Techniques: In Situ, Transduction

    Potential mechanisms and pathways involved in the effects of TGF-β in rAAV-transduced human normal and OA chondrocytes in situ . Explants were transduced with rAAV- lacZ or rAAV-hTGF-β as described in Figure 1 and maintained in culture for 90 days. The samples were then fixed and histologically processed for immunohistochemical detection of MMP-13 (A) , TIMP-1 (B) , TIMP-3 (C) , PTHrP (D) , β-catenin (E) , and the TGF-β receptor I (ALK1 and ALK5) (F and G , respectively ) (all at magnification x20; view of the middle zone).

    Journal: Journal of Translational Medicine

    Article Title: rAAV-mediated overexpression of TGF-? stably restructures human osteoarthritic articular cartilage in situ

    doi: 10.1186/1479-5876-11-211

    Figure Lengend Snippet: Potential mechanisms and pathways involved in the effects of TGF-β in rAAV-transduced human normal and OA chondrocytes in situ . Explants were transduced with rAAV- lacZ or rAAV-hTGF-β as described in Figure 1 and maintained in culture for 90 days. The samples were then fixed and histologically processed for immunohistochemical detection of MMP-13 (A) , TIMP-1 (B) , TIMP-3 (C) , PTHrP (D) , β-catenin (E) , and the TGF-β receptor I (ALK1 and ALK5) (F and G , respectively ) (all at magnification x20; view of the middle zone).

    Article Snippet: The anti-TGF-β (V), anti-MMP-13 (72B-01), anti-TIMP-1 (C-20) and -TIMP-3 (W-18), anti-parathyroid hormone-related protein (PTHrP) (1D1), anti-β-catenin (E-5), and anti-TGF-β receptor I (activin receptor-like kinase-1 ALK1: C-20; ALK5: T-19) antibodies were from Santa Cruz Biotechnology (Heidelberg, Germany).

    Techniques: In Situ, Transduction, Immunohistochemistry

    OvCa‐secreted TGF‐β transforms the pre‐metastatic peritoneum, favouring tumour progression. (A) Representative images of in vivo monitoring of SKOV3‐luc‐D3 cells in mice pre‐conditioned with TGF‐β1‐encoding adenovirus or control. Quantification of bioluminescence showed that tumour growth was increased in mice pre‐conditioned with TGF‐β1 adenovirus ( n = 6) as compared with control adenoviral pretreatment ( n = 6). (B) (a) Representative images of E‐cadherin immunostaining in the mesothelial monolayer of a mouse killed 2 days after being pre‐conditioned with conditioned medium (CM) from OvCa cells or control medium. Scale bars: 25 µm. (b) Diagram of experimental design. (c) Representative images of in vivo monitoring of SKOV3‐luc‐D3 cells and quantification of bioluminescence showed that intraperitoneal tumour growth was higher in mice pretreated with SKOV3 medium (CM). The TGF‐β receptor I inhibitor (GW) reduced tumour growth to levels comparable to those of mice whose peritoneums had not been pre‐conditioned (control medium). n = 6 per group. All mice were monitored for 41 days. Graphs represent mean average radiance (expressed as photons/s/cm 2 /sr) of SKOV3‐luc‐D3 cells ± standard error of the mean. Symbols represent the statistical differences over time between groups (** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). dpi, days post‐inoculation; i.p., intraperitoneal.

    Journal: The Journal of Pathology

    Article Title: Mesothelial‐to‐mesenchymal transition as a possible therapeutic target in peritoneal metastasis of ovarian cancer

    doi: 10.1002/path.4889

    Figure Lengend Snippet: OvCa‐secreted TGF‐β transforms the pre‐metastatic peritoneum, favouring tumour progression. (A) Representative images of in vivo monitoring of SKOV3‐luc‐D3 cells in mice pre‐conditioned with TGF‐β1‐encoding adenovirus or control. Quantification of bioluminescence showed that tumour growth was increased in mice pre‐conditioned with TGF‐β1 adenovirus ( n = 6) as compared with control adenoviral pretreatment ( n = 6). (B) (a) Representative images of E‐cadherin immunostaining in the mesothelial monolayer of a mouse killed 2 days after being pre‐conditioned with conditioned medium (CM) from OvCa cells or control medium. Scale bars: 25 µm. (b) Diagram of experimental design. (c) Representative images of in vivo monitoring of SKOV3‐luc‐D3 cells and quantification of bioluminescence showed that intraperitoneal tumour growth was higher in mice pretreated with SKOV3 medium (CM). The TGF‐β receptor I inhibitor (GW) reduced tumour growth to levels comparable to those of mice whose peritoneums had not been pre‐conditioned (control medium). n = 6 per group. All mice were monitored for 41 days. Graphs represent mean average radiance (expressed as photons/s/cm 2 /sr) of SKOV3‐luc‐D3 cells ± standard error of the mean. Symbols represent the statistical differences over time between groups (** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001). dpi, days post‐inoculation; i.p., intraperitoneal.

    Article Snippet: To assess the effects of interference with TGF‐β1 signalling, a total of 24 mice were treated with either a TGF‐β receptor I inhibitor (GW788388) (3 mg/kg per day) (Tocris Bioscience, Bristol, UK) or the vehicle dimethyl sulphoxide.

    Techniques: In Vivo, Mouse Assay, Immunostaining

    Differentiation of Th9 IL-4+IL-1β cells is transforming growth factor (TGF)-β independent. a – f T cells differentiation and gene array data are the same as shown in Fig. 2 . a Gene set enrichment analysis (GSEA) of Th9 IL-4+IL-1β cells versus classic Th9 IL-4+TGF-β cells or the Rest T helper (Th) cells for interleukin (IL)-1 signaling genes. Rest Th cells contain Th1, Th2, Th9 IL-4+TGF-β , and Th9 IL-4+TGF-β+IL-1β cells. b GSEA of Th9 IL-4+IL-1β versus classic Th9 IL-4+TGF-β cells or the Rest Th cells for TGF-β signaling genes. c , d Reverse transcriptase–PCR (RT-PCR) analysis of Il9 transcriptional level ( c ) and enzyme-linked immunosorbent assay (ELISA) of IL-9 production in the supernatants ( d ) from TGF-βRi-treated classic Th9 IL-4+TGF-β cells or Th9 IL-4+IL-1β cells at the indicated concentrations ( n = 3). TGF-βRi TGF-β receptor serine kinase inhibitor. e , f RT-PCR analysis of Il9 transcriptional level ( e ) and ELISA of IL-9 production in the supernatants ( f ) from αTGF-β-treated classic Th9 IL-4+TGF-β cells or Th9 IL-4+IL-1β cells at the indicated concentrations ( n = 3). αTGF-β TGF-β monoclonal antibody (mAb). g , h Naive CD4 + CD62L + T cells were purified from the spleens of wild-type (WT) mice or CD4dnTGF-βRII mice and cultured with plate-bound anti-CD3 mAbs and soluble anti-CD28 mAbs under polarized conditions, as detailed in the Methods section, for 3 days. RT-PCR analysis of Il9 transcriptional level ( g ) and ELISA of IL-9 production in the supernatants ( h ) of classic Th9 IL-4+TGF-β cells and Th9 IL-4+IL-1β cells from WT mice or CD4dnTGF-βRII mice after in vitro differentiation ( n = 3). CD4dnTGF-βRII mice mice expressing a dominant-negative TGF-β receptor. Data are mean ± SD; ** P

    Journal: Nature Communications

    Article Title: IL-4 together with IL-1β induces antitumor Th9 cell differentiation in the absence of TGF-β signaling

    doi: 10.1038/s41467-019-09401-9

    Figure Lengend Snippet: Differentiation of Th9 IL-4+IL-1β cells is transforming growth factor (TGF)-β independent. a – f T cells differentiation and gene array data are the same as shown in Fig. 2 . a Gene set enrichment analysis (GSEA) of Th9 IL-4+IL-1β cells versus classic Th9 IL-4+TGF-β cells or the Rest T helper (Th) cells for interleukin (IL)-1 signaling genes. Rest Th cells contain Th1, Th2, Th9 IL-4+TGF-β , and Th9 IL-4+TGF-β+IL-1β cells. b GSEA of Th9 IL-4+IL-1β versus classic Th9 IL-4+TGF-β cells or the Rest Th cells for TGF-β signaling genes. c , d Reverse transcriptase–PCR (RT-PCR) analysis of Il9 transcriptional level ( c ) and enzyme-linked immunosorbent assay (ELISA) of IL-9 production in the supernatants ( d ) from TGF-βRi-treated classic Th9 IL-4+TGF-β cells or Th9 IL-4+IL-1β cells at the indicated concentrations ( n = 3). TGF-βRi TGF-β receptor serine kinase inhibitor. e , f RT-PCR analysis of Il9 transcriptional level ( e ) and ELISA of IL-9 production in the supernatants ( f ) from αTGF-β-treated classic Th9 IL-4+TGF-β cells or Th9 IL-4+IL-1β cells at the indicated concentrations ( n = 3). αTGF-β TGF-β monoclonal antibody (mAb). g , h Naive CD4 + CD62L + T cells were purified from the spleens of wild-type (WT) mice or CD4dnTGF-βRII mice and cultured with plate-bound anti-CD3 mAbs and soluble anti-CD28 mAbs under polarized conditions, as detailed in the Methods section, for 3 days. RT-PCR analysis of Il9 transcriptional level ( g ) and ELISA of IL-9 production in the supernatants ( h ) of classic Th9 IL-4+TGF-β cells and Th9 IL-4+IL-1β cells from WT mice or CD4dnTGF-βRII mice after in vitro differentiation ( n = 3). CD4dnTGF-βRII mice mice expressing a dominant-negative TGF-β receptor. Data are mean ± SD; ** P

    Article Snippet: Reagents TGF-β receptor serine kinase inhibitor TGF-βRi (Cat#616452) was purchased from Millipore.

    Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Purification, Mouse Assay, Cell Culture, In Vitro, Expressing, Dominant Negative Mutation