In unstimulated endothelial cells, KDR is contained in an internal vesicular pool. (A) Unstimulated HUVECs and (B) unstimulated HMVECs stained for endogenous KDR (red). The boundaries of the cells are visualized by staining with the endothelial cell-cell adhesion molecule VE-cadherin (green). Nuclei are stained with DAPI (blue). (C) Analysis of the surface and intracellular pools of KDR in unstimulated HUVECs. Surface KDR was labeled with the membrane-impermeant biotinylation reagent sulfo-NHS-SS-biotin, as described in “Biochemical quantification of KDR trafficking.” Biotinylated surface KDR was collected by binding to streptavidin-agarose. Aliquots of the total cell lysate, surface fraction, and internal fraction were then analyzed by Western blotting with an anti-KDR antibody. No KDR was retrieved in the surface fraction in the absence of biotinylation. (D) Densitometric quantification of the relative surface and internal pools of KDR (mean ± SD; n = 3).