α4β1 integrin contributes to VEGF-induced signaling and function in CLL cells. (A) CLL cells (P13, P14, P15, P16, P17) were serum-starved for 2 hours and treated or not with the indicated inhibitors for 30 minutes at 37°C. Cells were added to wells coated with 5 μg/mL VEGF or 0.1% bovine serum albumin (BSA; control) and after 15 minutes at 37°C, cells were lysed and lysates analyzed by western blotting. VEGFR2 phosphorylation (p-VR2) at Y1214 is shown for a representative sample (P17) and quantitated for all 5 patients (P13, P14, P15, P16, P17) studied. (B) CLL cells (P3, P12, P15) were treated as in panel A and the phosphorylation of Akt and FAK (p-FAK) was analyzed by western blotting. (C) CLL cells (P10, P14, P15, P19, P20, P21, P22, P23) were treated with the indicated Abs for 30 minutes and incubated for 48 hours in the absence (blue bar) or presence (red bars) of immobilized VEGF (5 μg/mL). Cell viability was measured by the CCK8 method. The viability of cells treated with control Ab and VEGF was normalized to 1. Average values (arbitrary units [AU]) ± SEM are shown. *P < .05; **P ≤ .01; ***P ≤ .001.