Figure 5

Pak1 inhibitory domain (PID) reverts Rac inhibition of Rho by Rac. (A) HEK293T cells were transfected with plasmids expressing a GFP or GFP-PID fusion protein (PID) (1 μg/plate) and incubated for two days, serum-starved 4 h, and treated with thrombin (5 U/ml) for times indicated. Fresh lysates were used for Rho-pulldown assays with GST-Rhotekin to detect relative amounts of Rho-GTP. Total Rho levels were detected by western blot in total cell lysates as a loading control. GFP western blot from total cell lysate was used to detect the GFP-PID fusion protein. (A) Cells were co-transfected with plasmids encoding the active RacV12 (Rac) mutant in the presence of control pCEFL-GFP or pCEFL-GFP-PID (2 μg/plate). After 2 days incubation cells were serum-starved 4 h, stimulated with thrombin (5 U/ml, 2 min), and assayed for Rho-GTP levels with pulldown assays. Data are expressed as fold increase ± SEM in Rho-GTP with respect to unstimulated control cells.