Figure 5.

Requirements for βPIX and GIT1 in PAK-dependent permeability. (A) BAECs stimulated with 25 ng/ml VEGF for 30 min were fixed and immunostained for GIT1 or βPIX. (B) BAECs were left untreated or stimulated with VEGF and then extracted and immunoprecipitated with anti-phospho-S141 PAK antibody. The IPs were probes for phospho-MEK1/2, or, as a loading control, for phospho-PAK (pPAK). HC, IgG heavy chain. (C) BAECs were transfected by nucleoporation, using a protocol that gives 80–90% transfection efficiency, with WT PIX or ΔGBD PIX. Starved cells were untreated or stimulated with VEGF as described in A, extracted, and PIX immunoprecipitated. The presence of PIX and active MEK in the IPs was determined by Western blotting. (D) BAECs were nucleoporated with WT GIT1, GIT1 with a SPA2 homology domain (SHD) deletion that does not bind βPIX, WT βPIX, or ΔGBD βPIX. Cells were stimulated with VEGF as described in A and then fixed and stained for phospho-S141 PAK. (E and F) Cells were transfected and stimulated with VEGF as in A, then detergent extracted and analyzed by Western blotting to detect Erk phosphorylation (E) and MLC ser19 phosphorylation (F). (G) Quantitation. For Erk phosphorylation in E, values are means ± SE, n = 3, normalized for total proteins levels. Stimulation by VEGF was statistically significant (p < 0.01) as was the decrease in Erk activity after VEGF for GIT1 ΔSHD (p < 0.01) and PIX ΔGBD (p < 0.03) compared with vector control. For MLC, values are means ± range, n = 2, normalized to total protein levels. Stimulation by VEGF was statistically significant (p < 0.025) as was the decrease in Erk activity after VEGF for GIT1 ΔSHD and PIX ΔGBD compared with vector control (p < 0.04 for both).