Tgf β1

Manufactured by Promega

Sourced in United States

TGF-β1 is a recombinant human transforming growth factor-beta 1 protein. It is a multifunctional cytokine that regulates cell growth, cell proliferation, cell differentiation, and other cellular functions.

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Lab products found in correlation

Il 1β, by BD (3 mentions) Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions) Tgf β1, by R&D Systems (2 mentions) 96 well plate, by Corning (2 mentions) Tnf α, by Promega (1 mentions) Synergy ht luminometer, by Agilent Technologies (1 mentions) Prl tk plasmid, by Promega (1 mentions) Tnf α, by R&D Systems (1 mentions) Beas 2b, by American Type Culture Collection (1 mentions) Tgf β1, by Thermo Fisher Scientific (1 mentions) Mts assay, by Promega (1 mentions) Human tgf β1, by Thermo Fisher Scientific (1 mentions) Interleukin 6 (il 6), by Thermo Fisher Scientific (1 mentions) Ifn γ, by Thermo Fisher Scientific (1 mentions) Il 1β, by Thermo Fisher Scientific (1 mentions) Bovine ifn γ, by Kingfisher Biotech (1 mentions) Pcdna plasmid, by GenePharma (1 mentions) Dmem f12, by R&D Systems (1 mentions) Ly294002, by Promega (1 mentions) Fibroblast growth factor 2 (fgf2), by Promega (1 mentions)

15 protocols using tgf β1

Assessing NF-κB Transcriptional Activity

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For testing the NF-κB transcriptional activity, the 1 μg pGL6-NF-κB-Luc plasmid (Beyotime Institute Biotechnology, China) and 1 μg pRL-TK plasmid (Promega) as internal control were transiently co-transfected into LX-2 cells (1 × 10⁵ cells/well) using Lipofectamine 3000 (Invitrogen). After transfection for 12 h, cells were pre-treated with 10 μM NPLC0393, 10 μM SB431542, or 50 ng/mL NF-κB inhibitor SN50 for 12 h and were stimulated with 5 ng/mL TGF-β1 or 25 ng/mL TNF-α (R&D Systems, USA) for another 24 h.
For the measurement of MAT2A promoter activity, the Human MAT2A promoter-luciferase reporter plasmid containing binding sites for NF-κB (Sangon, China) and pRL-TK were transfected into LX-2 cells. After 12 h, cells were pre-treated with 10 μM NPLC0393 or 50 ng/mL SN50, a specific NF-κB translocation inhibitor, for 12 h and were stimulated with 5 ng/mL TGF-β1 or 25 ng/mL TNF-α for another 24 h. Dual luciferase assays were carried out according to the manufacturer's protocol (Promega) and light intensity was measured in a Synergy HT luminometer (BioTek, US). Each experiment was done in triplicate samples and results were normalized against those of cotransfected pRL-TK.

Wang K., Fang S., Liu Q., Gao J., Wang X., Zhu H., Zhu Z., Ji F., Wu J., Ma Y., Hu L., Shen X., Gao D., Zhu J., Liu P, & Zhou H. (2019). TGF-β1/p65/MAT2A pathway regulates liver fibrogenesis via intracellular SAM. EBioMedicine, 42, 458-469.

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Cytotoxicity of Single-Walled Carbon Nanotubes

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Semiconducting and metallic SWCNT stock solutions were first prepared in DI H₂O at 1 mg/mL. BEAS-2B and THP-1 cells were obtained from ATCC (Manassas, VA). 1×10⁴ BEAS-2B cells were cultured in 0.1 mL BEGM in 96-well plates at 37 °C. THP-1 cells were pretreated with 1 µg/mL phorbol 12-myristate acetate (PMA) overnight and primed with 10 ng/mL lipopolysaccharide (LPS). Aliquots of 3 × 10⁴ primed cells were cultured in 0.1 mL medium with carbon nanotubes in 96-well plates (Costar, Corning, NY, USA) at 37 °C for 24 h. In order to provide less aggregated tubes that can be suspended in biological aqueous media, all the SWCNT suspensions were freshly prepared by adding the stock solutions to BEGM or RPMI 1640 media at 12.5–100 µg/mL in the presence of BSA (0.6 mg/mL) and DPPC (0.01 mg/mL). After 24 h of culture, the supernatants were collected for the measurement of IL-1β (BD Biosciences, San Diego, CA) and TGF-β1 (Promega, Madison, WI) using ELISA kits according to manufacturer’s instructions. Concentrations are expressed as pg/mL.

Wang X., Mansukhani N.D., Guiney L.M., Lee J.H., Li R., Sun B., Liao Y.P., Chang C.H., Ji Z., Xia T., Hersam M.C, & Nel A.E. (2016). Toxicological Profiling of Highly-Purified Metallic and Semiconducting Single-Walled Carbon Nanotubes in the Rodent Lung and E. coli. ACS nano, 10(6), 6008-6019.

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Cell Proliferation and Clonogenic Assay

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The HNC SAS cell line was derived from poorly differentiated human squamous cell carcinoma of the tongue and cultured in DMEM medium with 10% FBS (Gibco BRL and Life Technologies). The HNC FaDu cell line was derived from a squamous cell carcinoma of the hypopharynx and cultured in RPMI medium with 10% FBS (Gibco BRL and Life Technologies). For cell proliferation assay, cells (3000 cells/100 μl) were seeded into a 96-well plate and treated with or without TGFβ1 (5 ng/ml, PeproTech). Cell growth was examined using MTS assay (Promega, Madison, WI, USA) at 24, 48, and 72 h. For clonogenic ability, cells (50 cells/3 ml) were seeded in a 6-well plate and treated with or without TGFβ1 (5 ng/ml) for 10 days. Colonies were fixed by methanol for 10 min and stained with 3% crystal violate for 30 min, and then we calculated the colony number.

Sun W.H., Chen Y.H., Lee H.H., Tang Y.W, & Sun K.H. (2022). PDK1- and PDK2-mediated metabolic reprogramming contributes to the TGFβ1-promoted stem-like properties in head and neck cancer. Cancer & Metabolism, 10, 23.

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Cytokine Quantification in Cell Cultures

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Recombinant proteins used were as follows, bovine IL-6, bovine IFN-γ (Kingfisher Biotech) and human TGF-β1 (Peprotech). Recombinant proteins were re-suspended in complete media, see above, prior to use. ELISAs were used to quantify IL-17 (KingFisher Biotech), TGF-β1 (Promega), IL-6, IFN-γ, and IL-1β (Thermo-Scientific).

Peckham R.K., Brill R., Foster D.S., Bowen A.L., Leigh J.A., Coffey T.J, & Flynn R.J. (2014). Two distinct populations of Bovine IL-17+ T-cells can be induced and WC1+IL-17+γδ T-cells are effective killers of protozoan parasites. Scientific Reports, 4, 5431.

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Cytokine Secretion in MWCNT-Exposed Macrophages

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After 24 h of culture, the supernatants of MWCNT-exposed THP-1 cells were collected for the measurement of IL-1β (BD Biosciences, San Diego, CA) and TGF-β1 (Promega, Madison, WI), using ELISA kits according to manufacturer’s instructions. Concentrations were expressed as pg/mL. For primary BMDMs, cells in 100 μL tissue culture medium were plated at the density of 5 × 10⁴ per well in a 96-well plate with the addition of LPS (500 ng mL⁻¹) for 5 h. The medium was replaced with fresh media and the primed cells treated with MWCNTs at the doses of 12.5, 25, 50 and 100 μg/mL in the presence of LPS (10 ng/mL) for 24 h. The supernatants of the exposed cells were collected for IL-1β assessment.

Wang X., Liao Y.P., Telesca D., Chang C.H., Xia T, & Nel A.E. (2017). The Genetic Heterogeneity among Different Mouse Strains Impacts the Lung Injury Potential of Multi-walled Carbon Nanotubes. Small (Weinheim an der Bergstrasse, Germany), 13(33), 10.1002/smll.201700776.

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Modulating HSC Dynamics via siRNA

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Primary HSCs were transfected with small interfering RNA (siRNA), pcDNA plasmid or negative control (GenePharma, Suzhou, China) for 48 h using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA). The p66Shc siRNA sequence was sense 5'-GCA ACU UGA AGC UGG CCA ATT-3', antisense 5'-UUG GCC AGC UUC AAG UUG CTT-3'. The NLRP3 siRNA sequence was sense: 5'-GAU CCU AUU UGA AGA GUG U-3', antisense 5'-GAU CAA CCU CUC UAC CAG A-3'. Then cells were incubated with transforming growth factor-β1 (TGF-β1, 2 ng/ml for 24 h, Promega, USA) for different assays.

Zhao Y., Wang Z., Feng D., Zhao H., Lin M., Hu Y., Zhang N., Lv L., Gao Z., Zhai X., Tian X, & Yao J. (2019). p66Shc Contributes to Liver Fibrosis through the Regulation of Mitochondrial Reactive Oxygen Species. Theranostics, 9(5), 1510-1522.

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TGF-β1 and FGF2 Modulate EMT in RLE Cells

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Rat AEC2 cell line RLE-6TN (RLE) cells [48 (link)] were obtained from American Type Culture Collection (ATCC). RLE cells were cultured in Dulbecco’s-modified Eagle’s medium/Nutrient F-12 Ham (DMEM/F12) (Sigma) containing 10% FBS and subcultured every 2 d. To induce EMT, 1 10⁴ cells/cm² were cultured in DMEM/F12 containing 1% FBS for 24 h and then stimulated with 0.5 ng/ml recombinant human TGF-β1 (R&D Systems) dissolved in 4 mM HCl containing 0.1% bovine serum albumin for 48 h. To assess the effect of FGF2 on EMT, cells were stimulated with 0.5 ng/ml TGF-β1 and 10–100 ng/ml recombinant human FGF2 (Wako) with 100 μg/ml heparin. To analyze signaling pathways, cells were pretreated with 2–10 μM U0126 (Promega) or 10 μM LY294002 (Promega) for 30 min and then stimulated with 0.5 ng/ml TGF-β1 alone or in combination with 50 ng/ml FGF2.

Watanabe-Takano H., Takano K., Hatano M., Tokuhisa T, & Endo T. (2015). DA-Raf-Mediated Suppression of the Ras—ERK Pathway Is Essential for TGF-β1-Induced Epithelial—Mesenchymal Transition in Alveolar Epithelial Type 2 Cells. PLoS ONE, 10(5), e0127888.

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Cytokine Release in Nanomaterial Exposure

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BEAS-2B and THP-1 cells were obtained from ATCC (Manassas, VA). 1×10⁴ BEAS-2B cells were cultured in 0.1 mL BEGM in 96-well plates at 37 °C. THP-1 cells were pretreated with 1 µg/mL phorbol 12-myristate acetate (PMA) overnight and primed with 10 ng/mL lipopolysaccharide (LPS). Aliquots of 3 × 10⁴ primed cells were cultured in 0.1 mL medium with carbon nanoparticles in 96-well plates (Costar, Corning, NY, USA) at 37 °C for 24 h. All the carbon nanoparticle suspensions were freshly prepared. After 24 h of culture, the supernatants were collected for the measurement of IL-1β (BD Biosciences, San Diego, CA) and TGF-β1 (Promega, Madison, WI) using ELISA kits according to manufacturer's instructions. Concentrations were expressed as pg/mL.

Wang X., Duch M.C., Mansukhani N., Ji Z., Liao Y.P., Wang M., Zhang H., Sun B., Chang C.H., Li R., Lin S., Meng H., Xia T., Hersam M.C, & Nel A.E. (2015). Use of a Pro-Fibrogenic Mechanisms-Based Predictive Toxicological Approach for Tiered Testing and Decision Analysis of Carbonaceous Nanomaterials. ACS nano, 9(3), 3032-3043.

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Ocular Surface Wound Healing Biomarkers

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After completion of each scratch assay, 1.5 mL of supernatant was combined with 100 μL of protease inhibitor and stored at −80 °C until read for analysis. Concentrations of proteins related to ocular surface wound healing including EGF, platelet-derived growth factor (PDGF)-BB, and transforming growth factor (TGF)-β1 were measured for each group of each horse. Growth factors were measured using previously validated human enzyme-linked immunosorbent assay (ELISA) kits (EGF and PDGF-BB: R&D Systems, Minneapolis, MN, USA; TGF-β1: Promega, Madison, WI, USA) that crossreact with the horse.

Sherman A.B., Gilger B.C., Berglund A.K, & Schnabel L.V. (2017). Effect of bone marrow-derived mesenchymal stem cells and stem cell supernatant on equine corneal wound healing in vitro. Stem Cell Research & Therapy, 8, 120.

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Epithelial-Mesenchymal Transition Model

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Alveolar type Ⅱ epithelial (RLE-6TN) cells were obtained from the American Type Culture Collection (Manassas, VA, USA). Cells were cultured in Ham’s F12 medium (10% fetal bovine serum, FBS) at 37 ºC and 5% CO₂. The EMT cell model was generated by exposing RLE-6TN cells in Ham’s F12 medium (10% FBS) containing 10 ng/ml TGF-β1 (Promega, Madison, WI) for 48 h as previously described [28 (link)]. ASP (98% purity) was purchased from Yongye Bio-engineering Co., Ltd. (Shanghai, China) and used at 200 μg/mL for 12 h.

Qian W., Cai X., Qian Q., Wang D, & Zhang L. (2020). Angelica Sinensis Polysaccharide Suppresses Epithelial-Mesenchymal Transition and Pulmonary Fibrosis via a DANCR/AUF-1/FOXO3 Regulatory Axis. Aging and Disease, 11(1), 17-30.

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