Analysis of the requirements for VEGFR-2 and NRP-1 in VEGF-induced mitogenesis and signaling. (A) VEGF-induced proliferation in VEGFR-2 and NRP-1 silenced HUVECs. SiRNA-transfected cells (4000 cells/well) obtained 48 to 72 hours after transfection were cultured for 72 hours in culture medium supplemented with 18% heat-inactivated FBS and 25 ng/mL heparin with or without VEGF-A (25 ng/mL). ▪ indicates control siRNA; □, VEGFR-2 siRNA; and ▦, NRP-1 siRNA. Results represent the mean ± SD of 5 experiments, each performed in triplicate. (B) ERK activation in HUVECs after transfection of control, VEGFR-2, or NRP-1 siRNA evaluated by Western blotting with specific antibodies to phosphorylated (p) and total ERK. Cells obtained 48 hours after transfection were starved for 16 hours and then exposed for 10 minutes to 50 ng/mL VEGF-A.