Recombinant human tgf β

Manufactured by BioLegend

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Recombinant human TGF-β is a protein produced using recombinant DNA technology. It is a member of the transforming growth factor beta family of cytokines.

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TGF-β (94 citations) Human-TGF-β (3 citations)

9 protocols using recombinant human tgf β

Recombinant Protein-Mediated Cell Signaling

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Recombinant human TGF-β, recombinant FGF-2 and recombinant human TNF-α were purchased from Biolegend (San Diego, CA). Rapamycin was from ENZO life science (Loerrach, Germany). Bafilolycin A1 was from Invivogen. FITC-PHA was from Vector (FL-1111). FITC-PNA was from Sigma (L7381). DAPI was from Sigma (D9542). Alexa Flour 488, 594 or 647 conjugated anti-mouse or anti-rabbit IgG were from Jackson ImmunoResearch Laboratories (West Grove, PA). The primary antibodies against JLP (ab12331, 1:1000), Fsp-1 (ab197896, 1:200), α-SMA (ab124964, 1:1000), Fibronectin (ab45688, 1:500), Collagen-I (ab34710, 1:1000), Ki67 (ab16667, 1:250), TGF-β (ab92486, 1:500), phospho-Smad2 (ab188334, 1:1000), phospho-Smad3 (ab52903, 1:1000) and p62 (ab56416, 1:200) were all from Abcam (Cambridge, MA). Anti-LC3 antibody (ab51520, 1:100, Abcam) was used for immuno-staining and Anti-LC3 antibody (L7543, 1:1000, Sigma) was used for western blotting, respectively. Anti-Nephrin and anti-F4/80 (123120, 1:100) antibodies were from Progen and Biolegend, respectively. Anti-Caspase-3 (#9664, 1:1000), anti-Beclin 1 (#3738,1:1000) was from Cell signaling Technology (Danvers, MA). Anti-GAPDH (sc-365062, 1:2500) was purchased from Santa Cruz (Santa Cruz, CA).

Yan Q., Zhu K., Zhang L., Fu Q., Chen Z., Liu S., Fu D., Nakazato R., Yoshioka K., Diao B., Ding G., Li X, & Wang H. (2020). A negative feedback loop between JNK-associated leucine zipper protein and TGF-β1 regulates kidney fibrosis. Communications Biology, 3, 288.

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Cytokine and Adhesion Molecule Assay

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Recombinant mouse IL-23, E-selectin and P-selectin Fc-chimeras were from R&D Systems (Minneapolis, MN). Recombinant mouse IL-12, IL-2, IL-6, TNF-α, recombinant human TGF-β, and the following antibodies to mouse cytokines and adhesion molecules: IL-4 (clone 11B11), IFNγ (clone XMG 1.2), IL-2 (clone JES6-1A12), CD4 (clone GK 1.5), CD3 (clone145-2C11), CD28 (clone 37.51), IL-17A (clone 2C11-18H10.1), CD43 activation-associated glycoform (clone 1B11), and CD44 (clone IM7) are all from Biolegend (San Diego, CA). Anti- PSGL-1 and mouse TNF-α were purchased from BD-Pharmingen (San Jose, CA), and carrier free CCL20 from Peprotech (Rocky Hill, NJ). PMA and ionomycin were from SIGMA (St. Louis, MO). Secondary Abs coupled to alkaline phosphatase were from Promega (Madison, WI). Vibrant CFSE and Alexa 680 were from Life Technologies (Carlsbad, CA). Myelin Oligodendrocyte glycoprotein was purchased from Anaspec (Fremont, CA) and Pertussis Toxin was purchased from List Biological Laboratories (Campbell, CA). Anti-E-selectin (clone 9A9) antibody was generously provided by Dr. F. William Luscinskas (Brigham and Women's Hospital, Boston, MA) and IgG control was from Biolegend (San Diego, CA).

Velázquez F., Grodecki-Pena A., Knapp A., Salvador A.M., Nevers T., Croce K, & Alcaide P. (2015). CD43 functions as an E-selectin ligand for Th17 cells in vitro and is required for rolling on the vascular endothelium and Th17 cell recruitment during inflammation in vivo. Journal of immunology (Baltimore, Md. : 1950), 196(3), 1305-1316.

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Immune Modulation Protocol

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Recombinant human TGF-β, IFN-γ, IL-4, G M-CSF and M-CSF were purchased from BioLegend. Phorbol 12-Myristate 13-Acetate (PMA) (Millipore, USA) and PI3K inhibitor, LY294002 (Cell Signaling Technology, USA) were dissolved in dimethyl sulfoxide (DMSO) and kept at −80 °C until use. FuGene®HD transfection reagent (Roche, USA) was used for plasmid transfection and puromycin (Thermo Fisher Scientific, USA) was used to select transduced cells. Nivolumab (Opdivo®) (Bristol-Myers Squibb, USA) was used to block PD-1/PD-L1 interaction in vitro.

Sri-ngern-ngam K., Keawvilai P., Pisitkun T, & Palaga T. (2022). Upregulation of programmed cell death 1 by interferon gamma and its biological functions in human monocytes. Biochemistry and Biophysics Reports, 32, 101369.

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Generation of Allergen-specific Th9 Cells

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Allergen-specific Th9 cells were prepared as described previously [7 (link)8 (link)]. Briefly, ovalbumin (OVA)-specific naïve CD4⁺ T cells were isolated from splenocytes of DO11.10/RAG2^-/- mice by positive selection using EasySep Mouse CD4⁺ T Cell Isolation Kit (Veritas, Santa Clara, CA, USA). Cells were cultured with x-ray-irradiated splenocytes in AIM-V medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum. At the start of culture, 0.3-μM synthetic OVA323-339 peptide (Scrum Inc., Tokyo, Japan), 20-U/mL recombinant IL-2 (PeproTech, Rocky Hill, NJ, USA), 10-U/mL recombinant IL-4 (PeproTech), 5-ng/mL recombinant human TGF-β (BioLegend, San Diego, CA, USA), and 10-μg/mL anti-interferon-γ monoclonal antibody (R4-6A2, eBioscience, San Diego, CA, USA) were added. Seven days after the stimulation, cells were harvested and used for the adoptive transfer. The successful differentiation of Th9 cells was confirmed elsewhere [7 (link)8 (link)].

Koyama T., Miura K., Yamasaki N., Ogata S., Ito D., Saeki M., Hiroi T., Mori A, & Kaminuma O. (2021). Suppressive effect of dexamethasone on murine Th9 cell-mediated nasal eosinophilic inflammation. Asia Pacific Allergy, 11(3), e25.

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Recombinant TGF-β Preparation

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Recombinant human TGF-β (BioLegend) was prepared in PBS at 5 μg/ml for a final concentration of 5 ng/ml.

Lee H.N., Mitra M., Bosompra O., Corney D.C., Johnson E.L., Rashed N., Ho L.D, & Coller H.A. (2018). RECK isoforms have opposing effects on cell migration. Molecular Biology of the Cell, 29(15), 1825-1838.

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Evaluating TGF-β, FGF-2, and TNF-α effects on HK-2 cells

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Human TEC cell lines HK-2 was purchased from China Centre for Type Culture Collection and was maintained as previously described. Recombinant human TGF-β, recombinant FGF-2 and recombinant human TNF-α were purchased from Biolegend (San Diego, CA). HK-2 cells were treated with TGF-β or recombinant FGF-2 or recombinant human TNF-α for 24 h, respectively, and then were harvested and analyzed by qPCR and western blotting. Jlp siRNA and control siRNA was purchased from Qiagen (Germany). The siRNA transfection was carried out using HiPerFect transfection (Qiagen) according to customer’s protocol. The efficiency of transfection was assessed by the protein expression. The sequence used for knockdown of Jlp in this study was: 5′-CAGACCCGAGTGGAATCTTTA-3′. Bafilolycin A1 was from Invivogen. HK-2 cells treated with or without Bafilolycin A1, as well as the cells transfected with Jlp siRNA for 24 h,

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Th1 and Th17 Cell Polarization

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Addition of αCD44 or CTLA-4-Ig is specified in the figures (both BD Biosciences). In experiments where αCD44 was added, results are displayed as ratio of cytokine production in αCD44-treated and untreated cultures. For Th1 cell polarization 10 ng/ml murine recombinant IL-12 (Peprotech, Hamburg, Germany) and 10 μg/ml αIL-4 (clone: 11-B11, DRFZ, Berlin Germany) was added. For Th17 cell polarization 20 ng/ml murine recombinant IL-6 (Peprotech), 10 ng/ml murine recombinant IL-23 (Peprotech), 1 ng/ml human recombinant TGF-β, 10 μg/ml αIL-4 and αIFN-γ (clone: R4-6A2 and XMG1.2, BioLegend, San Diego, USA) was added.

Schumann J., Stanko K., Schliesser U., Appelt C, & Sawitzki B. (2015). Differences in CD44 Surface Expression Levels and Function Discriminates IL-17 and IFN-γ Producing Helper T Cells. PLoS ONE, 10(7), e0132479.

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Th1, Th17, and iTreg Cell Induction

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Th1 and Th17 cells were induced as previously described (31 (link)). In brief, 1 ×10⁵ naïve CD4 cells were activated with anti-CD3 (145-2C11, Biolegend) and anti-CD28 (37.51, Biolegend) antibodies in presence of 50 ng/ml mouse recombinant IL-12 or 5 ng/ml of human recombinant TGFβ (Biolegend) and 25 ng/ml mouse recombinant IL-6 (Peprotech) for 3 days. For iTreg induction cells were cultured with 50 ng/ml of human recombinant TGFβ with 100 IU/ml recombinant IL-2 (Chiron), as previously described (32 (link)). For T cell proliferation cells were stained with 5 uM Cell Trace Violet (Thermo Fisher Scientific) in PBS for 15 min at RT, washed with cell culture media, counted and plated as mentioned earlier. Cells were cultured in RPMI 1640 media containing 2.05 mM L-Glutamine, 10% FBS and 1 × penicillin/streptomycin (GE Healthcare). For some experiments MAPK Kinase Inhibitor PD98059 (Sigma Aldrich) was used.

Koliesnik I.O., Kuipers H.F., Medina C.O., Zihsler S., Liu D., Van Belleghem J.D, & Bollyky P.L. (2020). The Heparan Sulfate Mimetic PG545 Modulates T Cell Responses and Prevents Delayed-Type Hypersensitivity. Frontiers in Immunology, 11, 132.

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Immune Cell Isolation and Stimulation

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Lithium heparin vacutainers were purchased from BD Biosciences (San Jose, CA, USA); Tissue culture medium RPMI, fetal bovine serum (FBS), penicillin-streptomycin and phosphate buffered saline (PBS) from Gibco, Thermo Fisher Scientific (Dublin, Ireland); Concanavalin A (Con A) (Cat# sc-203007A), XTT tetrazolium sodium salt (CAS 111072-31-2) (Cat# sc-258336), harpagoside (CAS number: 19210-12-9) (Cat# sc-203073) and sparteine sulfate (CAS number: 299-39-8) (Cat# sc-471860) from Santa Cruz Biotechnology (Santa Cruz, CA, USA); human recombinant TGF-β (Cat# 580702) from BioLegend (San Diego, CA, USA); Mitomycin C (Cat# M4287) and Histopaque^®-1077(Cat# 10771) from Sigma-Aldrich (St. Louis, MO, USA).

Atwany N.Z., Hashemi S.K., Jayakumar M.N., Nagarkatti M., Nagarkatti P, & Hassuneh M.R. (2020). Induction of CD4+CD25+ Regulatory T Cells from In Vitro Grown Human Mononuclear Cells by Sparteine Sulfate and Harpagoside. Biology, 9(8), 211.

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