VEGF stimulation provokes recycling of KDR to the cell surface. HUVECs were stimulated with 100 ng/mL VEGF for 30 minutes and then fixed and stained (green) for F-actin (C) and endogenous KDR (B; red). Nuclei are stained with DAPI (blue). Panel A shows the merged image. In panel B, peripheral clusters of KDR⁺ vesicles (arrow) and a KDR-enriched membrane protrusion (arrowhead) are indicated. (D) HUVECs were stimulated with 100 ng/mL VEGF for 30 minutes and then fixed and stained for tubulin (green) and endogenous KDR (red). Peripheral KDR⁺ vesicles can been seen aligned with microtubules directly beneath the cell surface. Bar represents 10 μm. (E) HUVECs were surface biotinylated and then incubated in the presence (•) or absence (○) of 50 ng/mL VEGF. At the time points indicated, biotin on surface KDR was cleaved by incubation with MesNa, and then the remaining biotinylated KDR (internalized) was quantified by ELISA, as described in “Biochemical quantification of KDR trafficking.” Data represent the mean ± SEM of 3 independent experiments. (F) A similar assay was used to measure the rates of KDR recycling over a 35-minute time course (20 min internalization, 15 min recycling), as detailed in “Biochemical quantification of KDR trafficking.” Cells were treated with (▪) or without (□) 50 ng/mL VEGF. VEGF stimulation caused a significant increase in the rate of KDR recycling (P < .001). Data represent the mean ± SEM of 5 independent experiments.