p21/p/CAF regulates TGFβ transcriptional activity and Smad3 occupancy on SBE. A, SCP2 and SUM159 cells were treated with TGFβ. Cell lysates were analyzed by co-immunoprecipitation using specific antibodies, as indicated. B, HEK293 cells were co-transfected with myc-Smad2, myc-Smad3 and flag-p21 with or without p/CAF siRNA. Transfected cells were stimulated with TGFβ for eight hours. Cell lysates were immunoprecipitated with an anti-flag antibody and analyzed by immunoblotting using Smad2/3 and flag antibodies. C and D, SUM159 cells were transfected with Scr or p/CAF siRNAs as well as a flag-tagged p21 cDNA, treated with or without TGFβ. The mRNA levels of indicated genes were analyzed by real-time PCR (error bars indicate SEM; n = 3 independent experiments). E, SCP2 cells transfected with Scr and p/CAF siRNA were stimulated with or without TGFβ. Cell invasion was quantified by relative TGFβ fold induction (error bars indicate SEM; n = 3 independent experiments). F, Transfected SCP2 cells were subjected to immunoblotting p/CAF and β-tubulin. G, HEK293 cells were co-transfected with myc-Smad3, myc-Smad2 and p/CAF. Immunoprecipitated Smad2/3 using an anti-myc antibody was subjected to Western blotting. H, DNA precipitation (DNA IP) was performed using biotinylated control and 4× CAGA SBE oligonucleotides, following by streptavidin precipitation. Western blotting of Smad3 and p/CAF is shown. I, SCP2 cells were transfected with p21 or p/CAF siRNAs. Samples were subjected to DNA IP and immunoblotting of Smad3. J, Transfected SCP2 cells were stimulated with or without TGFβ for 16 hrs. Luciferase activity of CAGA12-luc was measured and normalized to β-galactosidase (error bars indicated SEM; n =3 independent experiments).