Smad2 is involved in TGF-β1-inhibited aromatase transcription. A) TGF-β1 induced Smad2 phosphorylation. Cells were treated with TGF-β1 (1 ng/ml) for 1 h and Smad2 and Smad3 phosphorylation was determined by Western blot analysis using specific antibodies. β-actin was used for the loading control. B) TGF-β1 induced Smad2 nuclear translocation. Cells were treated with TGF-β1 as above and cytoplamic and nuclear proteins were analyzed for the phosphorylated Smad2 levels. C) Validation of Smad2 siRNA. Cells were transfected with 2 μg of pSuper empty vector or Smad2 siRNA (siSmad2) cloned in pSuper. At various time points after transfection, cell lysates were prepared and probed for Smad2. D) Smad2 siRNA reversed the TGF-β1-inhibited aromatase transcription. Cells were transfected with pSuper or pSuper carrying Smad2 siRNA. Smad2 siRNA blocked the effect of TGF-β1 on aromatase transcription. Knockdown of Smad2 by its siRNA was confirmed by Western blotting.