Naïve CD4+ T cells were isolated from the spleens of WT mice or AhR-/- mice using the Naive CD4+ T-cell isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany). For Treg cell polarization, naive CD4+ T cells were stimulated with immobilized anti-CD3 mAb (2 μg/mL, clone145-2G1, eBioscience) and soluble anti-CD28 mAb (1 μg/mL, clone37.51, eBioscience) supplemented with human TGF-β1 (2.5 ng/mL, R&D Systems) and mouse IL2 (10 ng/mL, R&D Systems) with the indicated concentration of mesalamine for 3 days. For Th1 or Th17 cell polarization, naive CD4+ T cells were stimulated with immobilized anti-CD3 mAb and soluble anti-CD28 mAb for Th0; with recombinant mouse IL12 (10 ng/mL, R&D Systems) plus anti-IL4 mAb (10 μg/mL, clone11B11, eBioscience) for Th1; and with human TGF-β1, mouse IL6 (30 ng/mL, R&D Systems), anti-IL4 mAb, and anti-IFN-γ mAb (10 μg/mL, cloneXMG1.2, eBioscience) for Th17, with the indicated concentration of mesalamine, for 3 days.
Human tgf β1
Manufactured by R&D Systems
Sourced in United States, United Kingdom, Germany
Human TGF-β1 is a recombinant human protein that corresponds to the mature form of transforming growth factor beta 1. It is a multifunctional cytokine that regulates cell growth, differentiation, and other functions in many cell types.
Automatically generated - may contain errors
Lab products found in correlation
Mouse il 6, by R&D Systems (6 mentions)
Mouse il 2, by R&D Systems (4 mentions)
Anti il 4 neutralizing antibody, by Thermo Fisher Scientific (3 mentions)
Anti ifnγ neutralizing antibody, by Thermo Fisher Scientific (3 mentions)
Anti il 12 23 p40 neutralizing antibody, by Thermo Fisher Scientific (3 mentions)
Soluble anti cd28, by Thermo Fisher Scientific (3 mentions)
Anti cd3, by Thermo Fisher Scientific (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (3 mentions)
Sb431542, by Merck Group (3 mentions)
Polyclonal antibodies, by Thermo Fisher Scientific (2 mentions)
Fluorescent 700 or 800 secondary antibodies, by Thermo Fisher Scientific (2 mentions)
Fsp1 antibody, by Agilent Technologies (2 mentions)
Anti gfp monoclonal antibody, by Thermo Fisher Scientific (2 mentions)
Tgf β1, by Santa Cruz Biotechnology (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Duoset elisa, by R&D Systems (2 mentions)
75 protocols using human tgf β1
1
Differentiation of Naive CD4+ T Cells
2
Differentiation of Naive T Cells into Th Subsets
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CD4+ CD25−CD62LhiCD44lo naive T cells were enriched from spleen using the naive CD4 T cells isolation kit (Miltenyi Biotec) with more than 92% purity. Naive T cells were then plated and cultured in 24-well plates. Naive T cells (106/0.5 mL) were stimulated with plate-bound anti-CD3 (2.5 μg/mL; eBioscience), soluble anti-CD28 (3 μg/mL; eBioscience), and plate-bound recombinant DLL4 (1.65 μg/mL, R&D). In addition, recombinant cytokines and neutralizing antibodies were added to skew toward different Th cells in vitro. For Th1: mouse IL-12 (10 ng/mL), anti-IL-4 neutralizing antibody (10 μg/mL; eBioscience); for Th2: mouse IL-4 (10 ng/mL; R&D System), anti-IFNγ neutralizing antibody (10 μg/mL; eBioscience), anti-IL-12/23 p40 neutralizing antibody (10 μg/mL); for Th17 cells: mouse IL-6 (10 ng/mL; R&D System), human TGF-β1 (2 ng/mL; R&D System), anti-IFNγ neutralizing antibody (10 μg/mL; eBioscience), anti-IL-4 neutralizing antibody (10 μg/mL; eBioscience), and anti-IL-12/23 p40 neutralizing antibody (10 μg/mL; eBioscience) were added; for IL-27-inducing TR1, mouse IL-27 (20 ng/mL, R&D) were added; to skew toward in vitro-iTreg cells (iTreg), human TGFβ1 (2 ng/mL; R&D System) and mouse IL-2 (10 ng/mL; R&D System) were added at the same time.
3
Differentiation of Naive T Cells into Th Subsets
4
Isolation and Induction of Naïve T Cell Subsets
5
Establishing EGFR-Mutant Lung Cancer Cell Lines
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The EGFR-mutant human lung adenocarcinoma cell lines HCC827 and H1975 were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). The cells were seeded and grown in RPMI 1640 medium (Gibco) with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). PC-9 cells were obtained from Professor Cai-cun Zhou as a gift and were seeded in DMEM (HyClone, South Logan, UT, USA) supplemented with 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco). All cells were cultured in humidified incubators at 37 °C with 5% CO2.
Gefitinib and SIS3 were purchased from Selleck Chemicals (Houston, TX, USA). Human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). Mouse TGF-β1 was purchased from Novoprotein Scientific (Summit, NJ, USA).
Gefitinib and SIS3 were purchased from Selleck Chemicals (Houston, TX, USA). Human TGF-β1 was purchased from R&D Systems (Minneapolis, MN, USA). Mouse TGF-β1 was purchased from Novoprotein Scientific (Summit, NJ, USA).
6
Notch and TGF-β Signaling Pathway Analysis
7
Profibrotic Factors Regulation of Fibrosis
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Human EGF and bFGF were from Thermo Fisher Scientific (NY, USA). Human TGFβ1 and TNF-α were from R&D systems, Inc. (MN, USA). Human TGFβ2 and TGFβ3 were from Prospec-Tany TechnoGene Ltd. (East Brunswick, NJ, USA). Thrombin, PD98059, SB202190, SP600125 were purchased from Sigma-Aldrich Chemical Co. (St Louis, MO, USA). Human recombinant VEGF was purchased from Prospect Biotech (Rehovot, Israel). Estrogen and progesterone were purchased from Cayman Chemical Co. (Ann Arbor, Michigan, USA). Recombinant human CTGF was obtained from Thermo Fisher Scientific eBioscience (Waltham, MA, USA). The antibodies (Abs) raised against vimentin (sc-6260), HSP47 (sc-8352) and phospho-Smad2/3 (sc-11769) were from Santa Cruz Biotechnology Inc. (Dallas, TX, USA). The Ab for total Smad2/3 (#3102) was purchased from Cell Signaling Technology, Inc. (Danvers, Massachusetts, USA). The Ab for α-SMA (GTX100034) was purchased from GeneTex (Hsinchu City, Taiwan). The Abs for collagen (ab34710), CTGF (ab6992) and α-tubulin (ab7291) were purchased from Abcam (Cambridge, MA). TGFβ isoforms were dissolved in 4 mM HCl/0.1% BSA (vehicle).
8
Isolation and Culture of Mouse Aortic Endothelial Cells
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Mouse aortic endothelial cells (MAEC) were isolated as described (Kobayashi et al, 2005 (link)) and cultured in high-glucose DMEM (Sigma-Aldrich) supplemented with 10% FBS (HyClone, Logan, UT), 1 mM sodium pyruvate, 0.1 mM MEM Non-Essential Amino Acids, 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (Life Technologies, Zug, Switzerland). 1 × 105 cells/well were seeded into 24-well plates, cultured to confluence, and then stimulated with recombinant mouse VEGF-A164 or human TGF-β1 (R&D Systems, Abingdon, UK) at different concentrations (0, 1, 2.5, and 40 ng/ml) in DMEM with 0.5% FBS at 37°C. After 24 h of stimulation, cells were collected for RNA extraction (n = 4 samples/group).
9
Investigating Intracellular Signaling Pathways
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Recombinant bovine FGF2, human FGF9, human TGFβ1, mouse noggin/Fc chimera, human BMP4, human BMP6, and anti-BMP antibody (MAB3552) were from R&D Systems (Minneapolis, MN). The following antibodies were all purchased from Cell Signaling Technology (Danvers, MA): anti–phospho-p44/42 MAPK E10 mouse monoclonal (#9106), anti–total p44/42 MAPK (#9102), anti–phospho-p38 (#9211), anti–phospho-MEK1/2 (#9154), anti–phospho Raf-1 (#9427), anti–phospho-FRS2-α(Tyr-196) (#3864), and anti–phospho-Smad1 (Ser463/465)/Smad5 (Ser463/465) (#9511). Other antibodies used were as follows: for CP49, rabbit anti-mouse CP49 polyclonal serum (#899 or #900; both generous gifts of Paul FitzGerald, University of California, Davis, CA); for phospho-tyrosine, 4G10 (a kind gift from Brian Druker, Oregon Health and Science University, Portland, OR); for luciferase, #G745A from Promega (Madison, WI); for GFP, JL-8 from Clontech (Mountain View, CA); for phospho-Smad3, ab51451 from Abcam (Cambridge, MA); for total Smad 1/5, ab75273 from Abcam; for total Raf-1, sc-7267 from Santa Cruz Biotechnology (Santa Cruz, CA); for total p38, sc-535 from Santa Cruz; and for total MEK, M17030 from Transduction Labs (Lexington, KT). UO126 (used at 15 μM), PD173074 (100 nM), and dorsomorphin (5 μM) were from Calbiochem (La Jolla, CA). All other reagents, including TPA, were from Sigma-Aldrich (St. Louis, MO).
10
TGF-β1 Induced EMT Pathway Regulation
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Detailed information is available in Supplemental Materials and Methods.
Normal human lung fibroblasts (NHLFs) were purchased from Lonza (Walkersville, MD, USA). BEAS-2B cells and HEK293 cells were purchased from Korea Cell Line Bank (Seoul, Korea). Human TGF-β1 was obtained from R&D Systems (Minneapolis, MN, USA) and other chemicals were purchased from Sigma (St. Louis, MO, USA). Antibodies were purchased from the following companies: anti-E-cadherin, anti-zona occuludens 1 (ZO-1), anti-N-cadherin, anti-vimentin, anti-SMAD2, anti-SMAD3, anti-phospho-SMAD2 (Ser465/467), anti-phospho-SMAD3 (Ser423/425), anti-SMURF2, horse radish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG (Cell Signaling, Beverly, MA, USA), anti-TTC3, anti-hemagglutinin (HA), anti-DYK (FLAG), and anti-Myc antibodies (Sigma), anti-α-SMA antibody (Abcam, Cambridge, MA, USA), anti-ubiquitin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-GAPDH antibody (AbFrontier, Seoul, Korea). Anti-DYK (M2) and anti-Myc agarose beads were purchased from Sigma and Thermo Scientific (Rockford, IL, USA), respectively. Detailed information about antibodies was given in Supplemental Table1 . Reagents including E1 (UBE1), E2 (UBE2E3), and the reaction buffer for the in vitro ubiquitylation assay were purchased from Boston Biochem (Cambridge, MA, USA).
Normal human lung fibroblasts (NHLFs) were purchased from Lonza (Walkersville, MD, USA). BEAS-2B cells and HEK293 cells were purchased from Korea Cell Line Bank (Seoul, Korea). Human TGF-β1 was obtained from R&D Systems (Minneapolis, MN, USA) and other chemicals were purchased from Sigma (St. Louis, MO, USA). Antibodies were purchased from the following companies: anti-E-cadherin, anti-zona occuludens 1 (ZO-1), anti-N-cadherin, anti-vimentin, anti-SMAD2, anti-SMAD3, anti-phospho-SMAD2 (Ser465/467), anti-phospho-SMAD3 (Ser423/425), anti-SMURF2, horse radish peroxidase (HRP)-conjugated anti-rabbit IgG and anti-mouse IgG (Cell Signaling, Beverly, MA, USA), anti-TTC3, anti-hemagglutinin (HA), anti-DYK (FLAG), and anti-Myc antibodies (Sigma), anti-α-SMA antibody (Abcam, Cambridge, MA, USA), anti-ubiquitin antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and anti-GAPDH antibody (AbFrontier, Seoul, Korea). Anti-DYK (M2) and anti-Myc agarose beads were purchased from Sigma and Thermo Scientific (Rockford, IL, USA), respectively. Detailed information about antibodies was given in Supplemental Table
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