Regulation of PECAM-1 phosphorylation and binding activity by ICAM-1 and eNOS. (A) ICAM-1 crosslinking induced phosphorylation of PECAM-1. HUVECs were transfected with empty vector, WT ICAM-1, and Y518F mutant ICAM-1 cDNAs and subjected to crosslinking assay. The phosphorylation of PECAM-1 at Y686 was examined. (B) ICAM-1 crosslinking-induced PECAM-1 phosphorylation was inhibited by PP2 (10μM) and L-NAME (1mM) in HUVECs transfected with WT ICAM-1. (C) Phosphorylation of PECAM-1 was diminished in eNOS^−/− mouse lungs. (D) ICAM-1 crosslinking-induced increase in PECAM-1 binding activity. HUVECs transfected as above in 96-well plates were subjected to ICAM-1 crosslinking and PECAM-1 mAb antibody binding and cell-attached ELISA. After ICAM-1 crosslinking for 1 hour, the cells were subjected to the PECAM-1 mAb binding assay. The cells were washed and incubated with the PECAM-1 mAb diluted in medium (10 μg/mL) at 4°C for 1 hour. The cells were then fixed and incubated with HRP-labeled secondary Ab. Data are mean ± SD of triplicate samples from 1 of 3 individual experiments that yielded equivalent results. *P < .05 versus control.