sE-selectin–induced Src phosphorylation is inhibited by the Src inhibitor PP2.

HMVECs were stimulated with 50 nM sE-selectin for various time points as indicated (m indicates minutes; NS, nonstimulated). In the inhibition study, HMVECs were pretreated with PP2 (10 μM) for 2 hours prior to stimulating with sE-selectin, and PP2 was also present during the stimulation with sE-selectin. Cell extracts were prepared, and the protein content in each sample was quantitated. Samples (20 μg each) were resolved by 10% SDS-PAGE and probed with rabbit antihuman phospho-Src (*p-Src) antibody. To verify equal loading, the blots were stripped and reprobed with rabbit antihuman total Src antibody. (A) Representative blot from 3 independent experiments. (B) Ratio of p-Src to total Src band density ± SEM from 3 independent experiments. *Represents a significant difference (P < .05) between the respective groups. sE-selectin induced a marked increase in Src phosphorylation at 10, 20, and 30 minutes after stimulation. Pretreatment of HMVECs with PP2 significantly inhibited Src phosphorylation.