Figure 4. Oxidant stress-induced PECAM-1 phosphorylation stimulates interactions with NcK.

(A/B) Nck was immunoprecipitated from HAECs treated with H₂O₂, and Nck-interacting tyrosine phosphorylated proteins were identified by Western blotting with the 4G10 antibody. Enhanced interaction with a 130kDa tyrosine phosphorylated protein was blunted by PECAM-1 siRNA. Representative Western blots are shown. (C–E) PECAM-1 was immunoprecipitated from HAECs treated with H₂O₂. Changes in PECAM-1 phosphorylation (C/D) and Nck coimmunoprecipitation (C/E) were analyzed by Western blotting and normalized to total PECAM-1 in the immunoprecipitates. Representative Western blots are shown. (F) HAECs were treated as in (A) and PECAM-1/Nck interactions were analyzed in situ by proximity ligation assays. Average proximal ligations per cell is shown. n=5 independent experiments for A–E and n=4 independent experiments for (F). * p<0.05, ** p<0.01.