The effects of Y27632 or ROCK1-siRNA on EGF-induced phosphorylation of MEK1/2, p44/p42 MAP kinase, Akt and GSK-3β in Panc1 cells. (A) The cells were pretreated with 3 μM Y27632 or vehicle for 1 h, and then stimulated with 30 ng/ml of EGF or vehicle for the indicated periods. (B) The cells were transfected with 10 nM of a negative control siRNA or siRNAs specifically targeting ROCK1 (#1) 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 were performed with antibodies against phospho-MEK1/2, MEK1/2, phospho-p44/p42 MAP kinase, p44/p42 MAP kinase, phospho-Akt, Akt, phospho-GSK-3β, GSK-3β 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.