Peripheral KDR⁺ vesicles carry Src tyrosine kinase. HUVECs were stimulated with 100 ng/mL VEGF for 30 minutes and then fixed and stained for endogenous KDR (red). (A-C) Cells were transfected with GFP-Rab4. (D-F) Cells were costained for PECAM (green), which stained a broad ribbon beneath the sites of cell-cell contact. Clusters of peripheral KDR⁺ vesicles could be observed at the base of some of these structures (F; arrows); however, these KDR⁺ vesicles did not contain PECAM (D). (G-I) Cells were transfected with GFP-Src. Panels H and I show a magnified section of panel G (dashed square). Arrowheads indicate the positions of peripheral KDR⁺ vesicles that colocalize with Src. Nuclei are stained with DAPI (blue). Bar represents 10 μm. (J) HUVECs were surface biotinylated and then incubated in the presence or absence of 50 ng/mL VEGF or with VEGF and 1 μM AP23464. KDR recycling over a 35-minute time course (20 min internalization, 15 min recycling) was measured as described in “Biochemical quantification of KDR trafficking.” Data represent the mean ± SEM of 3 independent experiments. As for measurements of receptor internalization, VEGF stimulation significantly stimulated KDR recycling (P = .006), whereas addition of Src inhibitor reduced the VEGF stimulation back to the basal rate (P = .002).