CaM is required for thrombin-induced activation of Rac1 in CHRF-288-11 cells and EGF-induced activation of Rac1 in HeLa cells.

<p>(A) CHRF-288-11 cells or (B) HeLa cells were serum starved for 12 h and incubated with W7 (150 μM) for 10 min followed by addition of thrombin to CHRF-288-11 cells and EGF to HeLa cells for 1 min or 3 min. At the end of the incubation cells were lysed using RIPA buffer. After centrifugation, 60 µl of the supernatant was suspended in 20 µl 4X Laemmli's sample buffer to determine level of endogenous Rac1 in various samples by western blotting. The rest of the supernatant was incubated with GST-PAK1 for 2 h at 4°C. After incubation, the beads were washed three times with Rac1 washing buffer. The final bead pellet was suspended in 30 µl of Laemmli's sample buffer and heated at 100°C for 5 min. Western blotting was performed using mouse anti-Rac1 antibody. Quantification (adjusted for endogenous level of Rac1) was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different. #p<0.05 was considered significantly different compared with corresponding thrombin or EGF treatment. The experiments were repeated a minimum of three times. In part (C), an equal amount of lysate (500 µg) from HeLa cells transiently expressing wild type HA-Rac1 and stimulated for various times with EGF was incubated for 2 hrs at 4°C with anti-HA antibody coupled to agarose beads. At the end of incubation beads were washed and bound proteins analyzed using SDS-PAGE and western blotting using anti-CaM antibody. Quantification was carried out using Bio-Rad “quantity one” program and *p<0.05 were considered significantly different when compared to EGF at 0 min.