Deletion of RGT from the β3 cytoplasmic tail does not impair activation of endothelial cell αVβ3. (A) Immortalized lung endothelial cells were incubated for 30 minutes at room temperature with WOW-1 Fab in the presence or absence of 200 nM PDBu (to activate protein kinase C) or with 5 mM EDTA (to inhibit specific WOW-1 Fab binding). WOW-1 Fab binding was measured by flow cytometry as described in “Methods.” Data represent specific WOW-1 Fab binding, defined as binding inhibited by EDTA, and represent means ± SEM of 3 independent experiments. **P < .01; *P < .05; NS, not significant (paired Student t test). (B) Localization of activated αVβ3 at lamellipodial edges. Cells were plated onto coverslips coated with fibrinogen and allowed to spread for 1 hour in the presence or absence of 200 nM PDBu as indicated. Cells were fixed, permeabilized, and stained with WOW-1 Fab, with antibody to total β3, and with phalloidin for F-actin as described in “Methods.” Bar represent 30 μm.