The effects of Y27632 or ROCK1-siRNA on either EGF or TGF-α-induced phosphorylation of EGFR at tyrosine residues. (A-C) Panc1 (A), KP3 (B) and AsPc1 (C) cells were pretreated with the indicated concentrations of Y27632 or vehicle for 1 h, and then stimulated with 30 ng/ml of EGF or 30 ng/ml of TGF-α or vehicle for 5 min. The cell lysates were then harvested and Western blotting were performed with antibodies against phospho-EGFR (Tyr1045 and Tyr1068), EGFR and GAPDH. (D) Upper panels; Panc1 cells were transfected with 10 nM of a negative control siRNA or siRNAs specifically targeting ROCK1 (#1 and #2) for 48 h, and then stimulated with 30 ng/ml of EGF or vehicle for 5 min. The cell lysates were then harvested and Western blotting was performed with antibodies against ROCK1, phospho- EGFR (Tyr1068), EGFR and GAPDH. The intensities of protein bands in the Western blot analysis 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.