Plasmids pCDNA3.1-TGFBR2-WT (WT) and pCDNA3.1-TGFBR2-Mut (with variant p.Val538Ala) were constructed in Tsingke Biological Technology Company. Plasmid p3TP-Lux, a luciferase reporter construct containing a TGF-β responsive element driving luciferase expression was bought on addgene (#11767). When the confluency of HCT116 in six-well plate was about 60%, we cotransfected plasmid p3TP-Lux with WT or mutant TGFBR2 using Lipofectamine 3000 (Thermo Scientific). Twenty-four hours later, cells were treated with 5 ng/ml TGF-β1 (PeproTech, #100-21) in medium without serum for an additional 24 h. At last cells were collected to measure luciferase expression. Total protein concentration was used to control luciferase activity, and all conditions were normalized to unstimulated cells overexpressing WT TGFBR2. Four independent experiments were completed with t-tests indicating significance between conditions.
Tgfbr2
Manufactured by Thermo Fisher Scientific
TGFBR2 is a gene that encodes the transforming growth factor beta receptor type 2 protein. This protein is a transmembrane serine/threonine kinase receptor that binds to transforming growth factor beta (TGF-beta) ligands and plays a role in various cellular processes.
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Lab products found in correlation
Tgf β1, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Myecl imager, by Thermo Fisher Scientific (1 mentions)
P38 mapk, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Ecl advance western blotting detection kit, by GE Healthcare (1 mentions)
Phospho smad3, by Cell Signaling Technology (1 mentions)
β actin, by Merck Group (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Tgfbr1, by Thermo Fisher Scientific (1 mentions)
Smad3, by Cell Signaling Technology (1 mentions)
Phospho p38 mapk, by Cell Signaling Technology (1 mentions)
2 protocols using tgfbr2
1
TGF-β Receptor Mutant Luciferase Assay
2
Western Blot Analysis of NLRP3 and TGF-β Signaling
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A549 cells were lysed with RIPA buffer (Thermo Fisher Scientific) and protein concentration were determined using Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). After SDS-PAGE, protein samples were transferred to a PVDF membrane, and then the membrane was incubated with primary antibody for overnight at 4 °C. Primary antibodies we used in this study were following: NLRP3, phospho-Smad3 (Ser423/425), Smad3, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK (Cell Signaling Technology, Danvers, MA), SMAD7, TGFBR1, TGFBR2 (Thermo Fisher Scientific), and β-actin (Sigma). Following incubation with HRP conjugated secondary antibodies, the proteins were detected by ECL Advance Western blotting detection kit (GE health care) and myECLimager (Thermo Fisher Scientific). The signal intensity was analyzed by ImageJ software.
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