NRP2 enhances the VEGFR-2 phosphorylation threshold induced by VEGF-A and VEGF-C in HMVECs. (A) Immunoblot analysis of HMVECs transfected with control or NRP2 siRNA (100 nM each). Thirty-six hours after transfection, cells were lysed, and 20 μg total protein extracts were submitted to Western blot analysis with the indicated antibody. (B) Level of VEGFR-2 phosphorylation induced by VEGF-A (100 ng/mL) or VEGF-C (300 ng/mL) in HMVECs transfected with control or NRP2 siRNA. Phosphorylation was quantified by ELISA as described in Figure 2. Data are plotted as mean ± SEM. The statistical analysis of differences in VEGF-induced responses between control siRNA and NRP2 siRNA-transfected cells was carried out using ANOVA (*P ≤ .05; ***P < .001). Similar results were obtained in 2 independent experiments with VEGF-A and 4 with VEGF-C.