p21 interacts with Smad3 and modulates TGFβ-induced transcriptional activity. A, HEK293 and SCP2 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21. Transfected cells were stimulated TGFβ for 8 hrs. Cell lysates were immunoprecipitated with an anti-flag antibody and analyzed by immunoblotting using Smad2/3 and p21 antibodies. B, Transfected MDA cells were immunoblotting by phospho-Smad3 (p-Smad3), Smad2/3 and β-tubulin antibodies in response to TGFβ for 30 minutes. C, SCP2 cells were co-transfected with either mock, Scr siRNA, p21 siRNA or flag-tagged p21 construct (p21 cDNA) and SBE promoter construct (CAGA12-luc). Transfected cells were stimulated with or without TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured and normalized to β-galactosidase (error bars indicate SEM; n = 3 independent experiments). D and E, SUM159 and SCP2 cells were treated with or without TGFβ for the indicated times. The mRNA levels of indicated genes were then analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments).