Tyrosine-phosphorylated VEGFR-2 always colocalizes with NRP-1 on stimulation with VEGF-A₁₆₅a. PAECs stably expressing VEGFR-2 and NRP-1 were stimulated for 10 (A-B) or 180 (C-D) minutes with VEGF-A₁₆₅a (A-C) or VEGF-A₁₆₅b (B-D). Receptor phosphorylated at Y1212 accumulated at the plasma membrane 10 minutes after stimulation (A). In addition, vesicles with phosphorylated VEGFR-2 and NRP-1 are visible (A-B arrows). Phosphorylated VEGFR-2 disappeared from the plasma membrane 180 minutes after stimulation (C) and was now observed in NRP-1–positive vesicles (C arrows). Most VEGFR-2/NRP-1 positive vesicles showed a pY1212 signal after stimulation with VEGF-A₁₆₅a (C). In contrast, many VEGFR-2/NRP-1 positive vesicles showed no pY1212 signal after stimulation with VEGF-A₁₆₅b (D arrowheads). Similar data were observed for VEGFR-2 phosphorylation at Y1175 (supplemental Figure 4). Scale bar: 20 μm (insets: 5 μm).