Figure 2
Specific inhibition of Rac1 expression mimics the effect of Smad2 knockdown on basal proliferation and enhances growth inhibition induced by exogenous TGF-β1. (A) PANC-1 cells were transfected with transfection agent alone (-), or with different amounts (as indicated) of either irrelevant control (Co) or Rac1-specific (Rac1) siRNAs for 3 h. After another 48 h in normal growth medium in the absence or presence of TGF-β1 cells were either counted (lower panel) or subjected to [3H]-thymidine incorporation assay (upper panel). The percentage of TGF-β1-induced growth inhibition relative to the respective untreated control is indicated below the bars. Data represent the mean ± standard deviation from six wells (thymidine incorporation) or 3 wells (cell numbers) processed in parallel. Shown is a representative experiment each from three independent experiments. Asterisks, p < 0.001. (B) A fraction of the cells from (A) were subjected to immunoblot (IB) analysis for Rac1 and β-actin as loading control (upper panel), or were stimulated with TGF-β1 for 15 min and subjected to immunoprecipitation (IP) of active Rac1 (lower panel). The active Rac1 was subsequently detected by immunoblotting (IB) with a Rac1 antibody. The same antibody was used with equal amounts of total cellular protein from the original samples to visualize total levels of Rac1.