Stretch-induced PECAM-1 phosphorylation in intact ECs. (A) Confluent BAECs cultured on an elastic substrate were stretched (25% elongation) for 5 min or left unstretched and stained with anti–protein phosphotyrosine (4G10). Tyrosine phosphorylated proteins were associated with interendothelial contacts, but this localization pattern became more prominent in stretched cells. Bipolar arrow indicates the direction of stretch. Bar, 20 μm. (B) Confluent or sparse BAECs cultured on an elastic substrate were subjected to stretch (25% elongation) for the times indicated or left unstretched. PECAM-1 phosphorylation was analyzed by immunoprecipitating PECAM-1 and immunoblotting with anti–PECAM-1 and 4G10. Although PECAM-1 phosphorylation increased in confluent cells, no increase was observed in sparse cultures. Black lines indicate that intervening lanes have been spliced out. (C) Quantification of immunoblotting results. Relative levels of PECAM-1 phosphorylation were determined by measuring the intensity of immunoblotted bands (see Materials and methods) and expressed relative to the phospho–PECAM-1 level in unstretched cells (mean ± SEM). Sample size: n = 6 for confluent samples, n = 3 for sparse samples. Student's t test was used to compare each time point with unstretched sample for each category. *, P = 0.0007, 0.0006, and 0.0003 for 5, 10, and 15 min, respectively. #, P = 0.0733 (confluent, stretched 2 min) and 0.5077 (sparse, stretched 10 min).