Fig 1.

TGF-β inhibits BMP-induced transcriptional responses in an ALK5-dependent manner. (A, B, and E to G) MDA-MB-231 BRE cells and MDA-MB-231 CAGA cells were induced with the ligands indicated and assayed for luciferase and Renilla activities after 8 h. (C) C2C12 and HaCaT cells were transiently transfected with BRE-luciferase and TK-Renilla. After 24 h, the C2C12 cells were induced with the ligands shown and luciferase/Renilla assays were performed. At 24 h after transfection, HaCaT cells were starved overnight in Opti-MEM before ligand induction. (D) MDA-MB-231 cells were transfected with ID2-luciferase and TK-Renilla for 24 h prior to ligand induction. In panel F, SB-431542 was added 15 min before the ligands shown, and in panel G, either dimethyl sulfoxide (DMSO) or SB-431542 was added 15, 30, or 60 min after ligand addition, as shown. All experiments were performed in triplicate. The data are presented as luciferase activity normalized to Renilla activity and are the mean and standard deviation from a single representative experiment. In panel E (right side), in parallel with the luciferase assay, MDA-MB-231 BRE cells were treated with ligands as indicated for 1 h. Whole-cell extracts were Western blotted using antibodies against PSmad1/5, PSmad2, and PSmad3, as well as total Smad1, -2, and -3. MCM7 is a loading control. The asterisk indicates a nonspecific band.