The effects of EGF on RhoA activity and the phosphorylation of cofilin, MLC and EGFR at tyrosine residues in Panc1 cells. (A) Panc1 cells were stimulated with 30 ng/ml of EGF for the indicated periods, and RhoA activity was measured by G-LISA™ Small G-protein Activation Assays. The results are expressed as the fold increase compared with untreated control cells. Bars designate SD of triplicate assays. (*) indicate significant increases (p < 0.05) compared to control cells. (B) Panc1 cells were stimulated with 30 ng/ml of EGF for the indicated periods. (C) Panc1 cells were pretreated with 3 μM Y27632 or vehicle for 1 h, and stimulated with 30 ng/ml of EGF or vehicle for 10 min. The cell lysates were then harvested and Western blotting were performed with antibodies against phospho-cofilin, cofilin, phospho-EGFR (Tyr1045 and Tyr1068), EGFR and GAPDH. The intensities of protein bands were determined by integrating the optical density over the band area using the NIH image software program. The intensity of each protein band was divided by the control (lane 1), and is shown above each panel. (D) Panc1 cells were treated with 3 μM Y27632 or vehicle for 1 h, followed by exposure to 30 ng/ml of EGF or vehicle for 10 min. After fixation, they were exposed to anti-phospho MLC antibodies, Alexa Fluor 488®-conjugated goat anti-rabbit IgG antibodies and DAPI. The cells were examined by fluorescence microscopy.