To measure insulin-like growth factor 1 (IGF-1) (Elh-IGF1, RayBiotech, GA, United States), transforming growth factor-β1 (TGF- β1, Enzo Life Science Inc, NY, United States), and dihydrotestosterone (ALPCO, NH, United States) levels in the cell lysate, enzyme-linked immunosorbent assay was used. All reaction and results were calculated after data were obtained with a microplate reader (BMG Biotech, Berlin, GmBH) at a suggested wavelength according to the manufacturer’s instruction.
Tgf β1
Manufactured by Enzo Life Sciences
Sourced in United States
TGF-β1 is a recombinant protein used in cell culture and biochemical applications. It is a member of the transforming growth factor beta family and plays a role in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Elh igf1, by RayBiotech (1 mentions)
Lipofectamine 3000 reagent, by Thermo Fisher Scientific (1 mentions)
Tgf β, by Enzo Life Sciences (1 mentions)
Tapi 2, by Enzo Life Sciences (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
Insulin, by Thermo Fisher Scientific (1 mentions)
Chrion 99021, by Cayman Chemical (1 mentions)
Angiopoietin 1, by Abcam (1 mentions)
C2 ceramide, by Enzo Life Sciences (1 mentions)
Sybr green pcr master mix, by Roche (1 mentions)
Deta nonoate, by Enzo Life Sciences (1 mentions)
Tgf β1, by Merck Group (1 mentions)
6 protocols using tgf β1
1
Quantifying Cellular Growth Factors
2
Synchronizing Cell Cycle for TGF-β Signaling
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The 786-O and A498 RCC cell lines were bought from ATCC in 2012. Prior to experiments, both cell lines were authenticated by STR profiling (Identicell, Denmark) on 12-06-2014, and were cultured in RPMI media supplemented with 10% FBS.
One day prior to transfection, 1 × 106 cells (786-O and A498) were seeded in a 10 cm plate. To synchronize the cell cycle, cells were starved by adding Opti-MEM and incubating at 37°C for 15 min. Transient transfection of the cell lines with 20 μg C-terminally hemagglutinin (HA)-tagged ALK5 (ALK5-HA) [17 (link)] or 20 μg pcDNA 3.1(+) (control) was done using Lipofectamine 3000 reagent (Life Technologies) according to manufacturer's protocol. Cells were collected the next day after stimulation with 10 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN, USA) for 30 minutes. For the kinetic experiment, cells (786-O and A498) stimulated with 10 ng/ml TGF-β1 were collected at 0 min, 30 min, 1 h, 2 h, and 6 h. For immunoblot experiments, the cells were treated with 10 ng/ml TGF-β or 10 ng/ml TGF-β and 20 μM TAPI-2 (Enzo life sciences, Farmingdale, NY, USA) or untreated cells were collected after 6 h.
One day prior to transfection, 1 × 106 cells (786-O and A498) were seeded in a 10 cm plate. To synchronize the cell cycle, cells were starved by adding Opti-MEM and incubating at 37°C for 15 min. Transient transfection of the cell lines with 20 μg C-terminally hemagglutinin (HA)-tagged ALK5 (ALK5-HA) [17 (link)] or 20 μg pcDNA 3.1(+) (control) was done using Lipofectamine 3000 reagent (Life Technologies) according to manufacturer's protocol. Cells were collected the next day after stimulation with 10 ng/ml TGF-β1 (R&D Systems, Minneapolis, MN, USA) for 30 minutes. For the kinetic experiment, cells (786-O and A498) stimulated with 10 ng/ml TGF-β1 were collected at 0 min, 30 min, 1 h, 2 h, and 6 h. For immunoblot experiments, the cells were treated with 10 ng/ml TGF-β or 10 ng/ml TGF-β and 20 μM TAPI-2 (Enzo life sciences, Farmingdale, NY, USA) or untreated cells were collected after 6 h.
3
Multilineage Differentiation of Clonal Cells
4
Platelet Secretion Factors ELISA
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At the end of the incubation of platelets with or without the agonists and after taking platelet samples for analysis in flow cytometry and microscopy, supernatants of the remaining platelets were collected after two centrifugation steps: 12 min, 1200 g at RT and 3 min, 13000 g at RT. Aliquots of supernatants were kept frozen at −80°C until analysis. The following ELISAs were used in accordance with the manufacturer’s instructions: serotonin (GenWay, San Diego, CA, USA), platelet factor 4 (PF4) (RayBiotech, Norcross, GA, USA), TGF-β1 (Enzo Life Science, Villeurbanne, France) and angiopoietin-1 (Abcam, Paris, France).
5
Evaluating Cellular Stress Responses
6
Keratocyte Regulation by TGFβ1 and Nitric Oxide
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Keratocytes were stimulated by TGFβ1 (catalog number T7039, Sigma-Aldrich Corp., St. Louis, MO, USA) (5 or 10 ng/mL) for 24 h. The expression of αSMA, N-cadherin, Smad3 and phosphorylated Smad 3 were evaluated by Western blot. For sodium nitrite treatment, various concentrations (0, 10, 100, 1000 μM) of sodium nitrite were added to the culture media after 24 h of TGFβ1 (10 ng/mL) stimulation. After additional 24 h of incubation with sodium nitrite, cells were harvested for further analysis. For DETA NONOate treatment, 10 and 100 μM of DETA NONOates (catalog number ALX-430-014, Enzo Life Science, Lausen, Switzerland) were added to the culture media after 24 h of TGFβ1 (10 ng/mL) stimulation. The expression level of αSMA was evaluated by Western blot.
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