Figure 2
Depletion of Rac1b increases constitutive and growth factor-induced ERK1/2 phosphorylation. (A) Panc1 cells were transiently transfected with siRNA specific to Rac1b (R1b) or irrelevant control siRNA (Co), serum-starved for 24 h and treated with TGF-β1 as indicated. Cells were subjected to immunoblotting for p-ERK1/2, t-ERK1/2 and HSP90 as well as Rac1b as a control for transfection efficiency. The chart below shows the relative intensities of the p-ERK1/2 bands normalized to those for t-ERK1/2 from three independent experiments (mean ± SD, n = 3). Data are displayed relative to non-stimulated Rac1b siRNA-transfected cells set arbitrarily at 1. Asterisks indicate a significant difference. (B) Colo357 and IMIM-PC1 cells were transiently transfected twice on two consecutive days with 50 nM each of a siRNA specific for Rac1b (R1b), or an irrelevant control siRNA (Co), serum-starved for 24 h and subjected to immunoblotting for phospho-ERK1/2 (p-ERK1/2) and total ERK1/2 (t-ERK1/2) as well al HSP90 as a loading control. The charts below the blots show the relative intensity of the p-ERK1/2 bands normalized to the t-ERK1/2 bands of three independent experiments (mean ± SD, n = 3). Asterisks indicate a significant difference. (C) Panc1 cells stably expressing a dominant negative ALK5 mutant (ALK5KR) or empty vector were transfected twice with 50 nM each of Co siRNA or Rac1b siRNA and subjected to immunoblot analysis of p-ERK1/2. The blot was stripped and reprobed with antibodies against and t-ERK1/2 and Rac1b. The graph below the blots show the relative intensities of p-ERK1/2 normalized to those for t-ERK1/2 from three independent experiments (mean ± SD, n = 3). (D) Panc1 cells were transfected with 50 nM of irrelevant Co siRNA, Rac1b siRNA or ALK5 siRNA, or 25 nM each of Rac1b + ALK5 siRNA. Cells were serum-starved, treated with TGF-β1 for 12 h and subjected to immunoblotting for p-ERK1/2, t-ERK1/2 and HSP90 as well as for Rac1b and ALK5 to verify successful depletion. The graph below the blots shows the relative intensities of p-ERK1/2 bands normalized to those for t-ERK1/2 from three independent experiments (mean ± SD, n = 3). Data are displayed relative to non-stimulated Rac1b siRNA-transfected cells set arbitrarily at 1. The asterisks indicate significance. Data displayed in each panel are from the same blot/gel treated subsequently with antibodies to p-ERK1/2, Rac1b, HSP90, and ALK5, then stripped and reincubated with an antibody to t-ERK1/2.
