Figure 3
Effects of ectopic overexpression of Rac1b on TGF-β1-induced ERK1/2 activation and gene expression. (A) Panc1 cells stably overexpressing Rac1b from the pCGN vector (two independent clones, #4 and #13) as well as empty-vector control cells (v) were serum-starved, treated with TGF-β1 for 12 h and subjected to immunoblotting for p-ERK1/2, t-ERK1/2, and HA-Rac1b. The graph underneath the blot shows results from densitometric analysis of the respective bands for p-ERK1/2 normalized to those for t-ERK1/2 and derived from four independent experiments (mean ± SD, n = 4). Data are displayed relative to TGF-β1 stimulated vector control cells set arbitrarily at 1. Asterisks indicate a significant difference of p-ERK1/2 band intensity between the vector control and the two HA-Rac1b expressing clones. Due to the short exposure time, endogenous Rac1b protein is not visible. (B) The same cells as in (A) were stimulated with TGF-β1 for 24 h and subjected to qPCR for E-cadherin and PAR2. Data are displayed as TGF-β1-treated over control cells and are the mean ± SD of 3 experiments. Asterisks indicate significant differences. Data are from the same blot/gel treated subsequently with antibodies to p-ERK1/2, Rac1b, and HSP90, then stripped and reincubated with an antibody to t-ERK1/2. The vertical lines between lanes 3 and 4 indicate removal of irrelevant lanes from the blot.
