VE-cadherin is internalized in clathrin coated-vesicles. (A) HUVEC were cultivated on collagen-coated slides, serum deprived, subjected to VE-cadherin internalization assay as described in  and stimulated with VEGF (50 ng/ml, 15 min). Cells were fixed and stained for VE-cadherin (iVEC, green), microtubule (red) and actin (purple). Cells were then analyzed by confocal microscopy. Scale bar: 10 μm. (B) VE-cadherin staining was performed as described above, analyzed by confocal microscopy and by 3D reconstitution using IMARIS software. Nuclei are presented in blue (DAPI) and iVEC in green. Scale bar: 10 μm. (C) Confocal analysis of iVEC (green), together with clathrin heavy chain (HC), adaptin α or rab5 (red) in HUVEC stimulated with VEGF (50 ng/ml, 15 min). Scale bar: 10 μm. (D-F) HUVEC received adaptin α-targeting (AP2si) and non-silencing (nsi) duplexes. Total cell lysates were analyzed by western-blot three days later for adaptin α and Tubulin protein expression levels. Confocal analysis of iVEC (green) was performed in the absence (Ctrl) or presence of VEGF (50 ng/ml, 15 min). Graph represents the mean ± s.e.m. of the percentage of cells with iVEC staining; n >300; T-test: ** p < 0.01. Scale bar: 10 μm. All panels are representative of at least 3 independent experiments.