VE-cadherin internalization assay and immunofluorescence

The internalization assay was performed at 48-h post-transfection as previously described [9]. Briefly, HUVECs were grown on coverslips to confluence before labeling the cell surface-exposed VE-cadherin by incubation with mouse anti-human VE-cadherin (Supplementary Table 3) at 4 °C for 1 h. Movement of labeled VE-cadherin was monitored by placing the cells at 37 °C for 1 h, in basal medium with or without VEGF (50 ng/ml; Preprotech), after which the cells were either washed with PBS (PBS supplemented with 1.8 mM CaCl2 and 1 mM MgCl2) or with a mildly acidic buffer [2 mM PBS-glycine (pH 2.0), 15 min] to remove membrane-bound antibodies and reveal the internalized labeled VE-cadherin. Cells were fixed with 4% paraformaldehyde for 30 min, followed by a blocking and permeabilization step for 30 min in PBS with 1% BSA and 0.5% Triton-X. Secondary antibody (Supplementary Table 3) incubation was performed for 1 h at room temperature, followed by a 30-min incubation step to label cytoskeletal F-actin with rhodamine phalloidin (Supplementary Table 3). For labeling of Rab proteins (Supplementary Table 3), an extra primary antibody step was performed after fixation instead of labeling cytoskeletal F-actin. DAPI was used as a nuclear counterstain. Images of fluorescent-labeled markers were obtained with a Leica TCS SP8 X microscope and a 63x oil immersion objective lens. 3D images were obtained by scanning multiple XY planes in the Z direction with a depth of ± 10 µm. Serial pictures along the Z-axis were combined to create a stacked XY image. The total internalized VE-cadherin area was determined by using ImageJ 1.47v.