Fig. 6. The effect on activation of Rho GTPases of inhibitors of p38 MAPK and phosphatidylinositol-3 kinase. (A) PC-3U/pMEP4-S7 were starved in 1% FBS for 12 hours, pretreated with or without either 10 µM SB203580 or 10 µM LY294002 for 1 hour, and stimulated with 1 µM of CdCl₂ for 12 hours. The parental cells, PC-3U, were stimulated with 5 ng/ml of TGF-ß for 12 hours as positive control and CdCl₂ for 12 hours as negative control. The amount of active, GTP-loaded Rac1, Cdc42 and RhoA was determined by GST pull-down assays with GST-PAK-CRIB, GST-WASP-CRIB or GST-Rhotekin, respectively. Rac1, Cdc42 and RhoA were detected by immunoblotting using antibodies specific for the respective GTPase. (B) Immunoblots were analyzed by densitometry and the data are combined into diagrams showing the activation of Rac1, Cdc42, and RhoA. Each column represents the mean + s.e.m. of three independent experiments.