NRP2/VEGFR-2 interaction enhances VEGFR-2 phosphorylation induced by VEGF-A and VEGF-C in PAECs. PAECs stably expressing VEGFR-2 were transfected with NRP2. (A) Western blot analysis of VEGFR-2 and NRP2 expression in VEGFR-2– and VEGFR-2/NRP-2–expressing cells. The blot was cut around 70 kDa; the top part was incubated with either anti–VEGFR-2 or anti-NRP2 and the bottom part with anti-Erk antibodies. (B-C) Phosphorylation of VEGFR-2 induced by VEGF-C in cells coexpressing NRP2 (C) or not (B). Cells were stimulated with the indicated concentration of VEGF-C for 10 minutes at 37°C. Cell lysates were immunoprecipitated with anti–VEGFR-2 antibodies. Samples from PAEC VEGFR-2 and PAEC VEGFR-2/NRP2 were run on 2 separate gels, but membranes were exposed together on the same film for the same amount of time. Proteins were immunoblotted with anti-phosphotyrosine (PY), anti–VEGFR-2, or anti-NRP2 (C). The values below the anti-NRP2 indicate the fold increase in the association between NRP2 and VEGFR-2 in response to VEGF-C. (D-E) Phosphorylation of VEGFR-2 was quantified by ELISA, and specific signal was normalized to the total amount of VEGFR-2 which was also measured by ELISA. Cells were stimulated with the indicated concentration of either VEGF-A or VEGF-C as described in panels B and C. Results are representative of 2 independent experiments, and data are shown as mean ± SEM.