Figure 4

The in vitro interaction of RAC1 with PAK1, a downstream effector. (a) Interaction with the p21-binding domain (PBD) of PAK1. COS7 cells were transfected with pCAG-Myc-RAC1, -Myc-RAC1-p.Tyr40His, -Myc-Trio-D1 (GEF), and -Myc-Luciferase (Luc) vectors (0.3 μg each) in various combinations. Pull-down assays were conducted with GST-fused PAK1-PBD (5 μg each). Bound RAC1 proteins were detected by western blotting with anti-Myc. Total cell lysates were also immunoblotted with anti-Myc for normalization (input). (b) Relative band intensities of precipitated PAK1 were normalized against WT and are shown as fold-increase over the WT level below the image. Asterisks indicate statistically significant differences as determined by Tukey’s test. GEF + WT vs. WT, p < 0.001; p.Tyr40His vs. WT, p = 0.525; GEF + p.Tyr40His vs. WT, p = 0.454; p.Tyr40His vs. GEF + WT, p < 0.001; GEF + p.Tyr40His vs. GEF + WT, p < 0.001; GEF + p.Tyr40His vs. p.Tyr40His, p = 0.999. (c) Activation of exogenous PAK1. COS7 cells were transfected with pCAG-Myc-PAK1 and pCAG-Myc-Luciferase (Luc) or -Myc-Trio-D1 (0.1 μg each) together with pCAG-Myc vector (–), -Myc-RAC1, and -Myc-RAC1-p.Tyr40His (0.3 μg each). After 24 h, cell lysates were prepared and subjected to western blotting. Activated Myc-PAK1 was detected with an anti-phospho-Ser199/204-PAK1 antibody (bottom panel). Protein levels of Myc-Trio-D1, Myc-PAK1, Myc-Luc, and Myc-RAC1 were confirmed with an anti-Myc antibody (upper and middle panels). (d) Relative band intensities of phosphorylated PAK1 were normalized based on the PAK1 level and quantified against the positive control without RAC1 (lane 1) and are analyzed and shown as in (b). WT vs. CTRL, p = 0.51505; p.Tyr40His vs. CTRL, p = 0.81731; GEF vs. CTRL, p = 0.00187; GEF + WT vs. CTRL, p < 0.001; GEF + p.Tyr40His vs. CTRL, p = 0.66883; p.Tyr40His vs. WT, p = 0.99266; GEF vs. WT, p = 0.03513; GEF + WT vs. WT, p < 0.001; GEF + p.Tyr40His vs. WT, p = 0.99975; GEF vs. p.Tyr40His, p = 0.01383; GEF + WT vs. GEF, p < 0.001; GEF + p.Tyr40His vs. p.Tyr40His, p = 0.99970; GEF + WT vs. p.Tyr40His, p = 0.01623; GEF + p.Tyr40His vs. GEF, p = 0.02235; GEF + p.Tyr40His vs. GEF + WT, p < 0.001. (b,d) Number of replicates, N ≥ 3. p-value was calculated as in (Fig. 3b). ***p < 0.001; **p < 0.01; *p < 0.05; n.s. not significant.