FIGURE 4.

FAK induces N-WASP phosphorylation that restores endothelial barrier function. A and B, time course of tyrosine phosphorylation of N-WASP following thrombin stimulation. HPAEC were stimulated with 50 nm thrombin for the indicated time points, after which lysates were either immunoprecipitated with N-WASP antibody and immunoblotted with total phospho-Tyr (pTyr) antibodies (A) or phospho-Tyr-256 (pTyr256) N-WASP antibody (B). The plot in A and B shows mean ± S.D. of increase in N-WASP phosphorylation from three individual experiments. * indicates significance from control unstimulated condition (p < 0.05). C, effect of FAK knockdown on N-WASP phosphorylation. HPAEC transfected with scrambled (siSc) or FAK siRNA (siFAK) were stimulated with 50 nm thrombin for 30 min. Cell lysates were electrophoresed and immunoblotted with FAK or N-WASP phospho-Tyr-256 antibodies. The plot represents mean ± S.D. of -fold increase in phosphorylation from three individual experiments. * indicates significance from scrambled siRNA transfected cells after thrombin treatment (p < 0.05). D, effect of N-WASP phosphorylation on endothelial barrier function. HPAEC plated on gold electrodes were transfected either with phosphospecific N-WASP mutants Y256D and Y256F or with GFP alone. Cells were stimulated with 50 nm thrombin, and changes in TER were determined. Data represent mean ± S.D. from three individual experiments performed in triplicates. * indicates significance from cells expressing Y256D mutant or control vector (GFP) (p < 0.05).