ICAM-1 activation-dependent stimulation of Src in HUVECs. (A) Mouse WT and phosphorylation-defective ICAM-1 mutant (Y518F) expressed in HUVECs were detected by ELISA. (B) Comparison of TNF-induced endogenous ICAM-1 expression and mouse ICAM-1 expression in transfected HUVECs. (C) ICAM-1 crosslinking (XL) was performed using a rat anti–mouse ICAM-1 mAb (YN1/1.7.4) against the mouse ICAM-1 extracellular LFA-1 binding domain. The expression of flag-tagged mouse WT and mutant ICAM-1 in human ECs was equivalent. Phosphorylation of Src at Y418 (human) and ICAM-1 at Y518 was determined using phospho-specific antibodies and Western blot analysis. Summarized results of protein phosphorylation assays are based on 3 or 4 experiments (mean ± SD). (D) HUVECs were cotransfected with mouse ICAM-1 and WT or phosphatase-dead C463S SHP2 mutant. Phosphorylation of Src Y419 (active site) increased and of Y530 (inactive site) decreased in the cells after ICAM-1 crosslinking. Phosphorylation of ICAM-1 Y518 was enhanced in the presence of WT SHP2. *P < .05 versus noncrosslinked control.