Fig 7.

The inhibitory effect of TGF-β on BMP transcriptional responses depends on Smad3 but not on Smad2. (A to D) MDA-MB-231 BRE cells were transfected with nontargeting (NT) siRNA or siRNA duplexes against Smad2 (S2), Smad3 (S3), or ALK5, as indicated. After 72 h cells were stimulated with the ligands shown and luciferase/Renilla assays were performed as in the legend to Fig. 1 (A and C) or whole-cell extracts were prepared and blotted for Smad2/3, Smad4, or ALK5 as indicated (B and D). In the case of ALK5, samples were treated with or without peptide N-glycosidase F (PNGase F) as described in the legend to Fig. 3. (E) MDA-MB-231 BRE cells were treated with NT or Smad3 siRNA for 72 h as described above and then induced with ligands for 1 h. Total RNA was prepared, and levels of ID2, relative to that of the gene for glyceraldehyde-3-phosphate dehydrogenase, were measured by qPCR. The qPCRs were performed in triplicate, and means and standard deviations are shown. Controls for knockdown are those shown in panel D.