Figure 7.

An inhibitory peptide blocks the interaction between PAK and PIX. (A) Sequence of the PIX SH3-blocking peptide and the mutated control. (B) Cell lysates were incubated with PIX-blocking (PIX) or control (Con) peptides immobilized on streptavidin beads. The beads were rinsed, and the bound proteins or whole cell lysates (WCL) or analyzed. Western blots were probed for the indicated SH3 domain-containing proteins. (C) BAECs were incubated with PIX-blocking or control peptide at 20 μg/ml for 1 h and then stimulated with VEGF for 30 min. Cells were rinsed, lysed, and βPIX immunoprecipitated. The IPs were analyzed by Western blotting for PIX as a control, or for PAK and phospho-PAK (pPAK) to detect coIP. (D) BAECs were incubated with peptides and stimulated with VEGF as described in C and then lysed and analyzed for Erk activation by Western blotting against phospho-Erk. Total Erk was analyzed to demonstrate equal loading. Values are means ± SE, n = 3, normalized to total protein. VEGF stimulation of Erk phosphorylation was statistically significant, as was the inhibition byGIT Δ SHD (p < 0.02). (E) Cells were incubated with PIX-blocking or control peptides as described in C, stimulated with bFGF for 60 min, and then stained for F-actin.