Plexin-A4 modulates VEGF-induced signaling mediated by the VEGFR-2 receptor. (A) HUVECs expressing a nontargeting shRNA (sh-control) or HUVECs in which plexin-A4 expression was silenced (sh-plexA4) were stimulated with VEGF (10 ng/mL) as described. Adherent cells were counted after 3 days. The average of 3 independent experiments each of which was done in triplicates is shown. (B) Spheroids (500 cells/spheroid) containing HUVECs expressing a control shRNA (sh-control) or HUVECs silenced for plexin-A4 expression (sh-plexA4) were seeded in collagen and stimulated to sprout with 50 ng/mL VEGF. Shown are representative pictures of sprouting spheroids taken after 24 hours. (C) The full-length human plexin-A4 cDNA was fused to a c-terminal V5 tag and coexpressed with the VEGFR-2 cDNA in PAE cells. The cells were lysed and immunoprecipitation was performed using anti-V5 antibodies or anti-HA antibodies used as a negative control. Precipitates were subjected to Western blot analysis using antibodies directed against VEGFR-2. Blots were then stripped and reprobed with an anti-V5 antibody. (D) HUVECs expressing a nontargeting shRNA (sh-control) or an shRNA targeting plexin-A4 (sh-plexA4) were incubated with or without VEGF (10 ng/mL) at room temperature as indicated. After 10 minutes the cells were lysed, and subjected to Western blot analysis using an antibody against the phosphorylated Y-1175 residue of VEGFR-2. Blots were then stripped and reprobed with an antibody directed against VEGFR-2. (E) Confluent 6-well dishes containing PAE cells expressing VEGFR-2 and either plexin-A4-V5 or control vector were stimulated with increasing concentrations of VEGF as indicated. Cell extracts were analyzed by Western blot using an antibody directed against the phosphorylated Y-1175 residue of VEGFR-2. The membrane was subsequently stripped and probed with an antibody directed against VEGFR-2 to assess total VEGFR-2 levels in the extracts. All the experiments were repeated independently at least 3 times with similar results.