DEP-1 and its C-terminal Y1311 and Y1320 are essential mediators of VEGF-dependent endothelial permeability. (A) DEP-1 promotes VEGF-dependent VE-cadherin phosphorylation. HUVECs were transfected with control (CTL) or DEP-1 siRNAs, serum-starved for 6 hours, and then stimulated with VEGF for the indicated times. The phosphorylation level of Src, VE-cadherin, and Vav2 was determined by immunoblotting with the indicated phospho-specific antibodies. Results are representative of at least 3 independent experiments. (B) DEP-1 is required for VEGF-induced loosening of cell-cell junctions. Control (CTL) and DEP-1–depleted cells were stimulated with VEGF (50 ng/mL) for 30 minutes and stained with β-catenin and anti–mouse Alexa 488 secondary antibodies. Bottom panel, RNAi-mediated decrease in DEP-1 expression levels. Results are representative of 3 independent experiments. (C) DEP-1 mediates VEGF-induced endothelial permeability. Control (CTL) and DEP-1–silenced cells were plated on collagen-coated inserts. Forty-six hours later, cells were serum-starved for 2 hours and then stimulated or not with VEGF (50 ng/mL) for 30 minutes in the presence of FITC-dextran (in the upper chamber). VEGF-induced permeability was measured by detecting the fluorescence emitted by FITC-dextran present in aliquots that were collected from the bottom chambers. Data are presented as fold changes over unstimulated control (CTL) cells. Assays were conducted in triplicates and results are representative of 4 independent experiments; *P < .05. (D) HUVECs transfected with pmT2 empty vector, WT DEP-1, the Y1311F/1320F (YY/FF), or C/S mutants were plated on collagen-coated inserts, serum-starved for 1 hour and then stimulated for 30 minutes with VEGF in the presence of FITC-dextran, as described in panel C. Data are presented as fold changes over stimulated control (pmT2) cells. Assays were conducted in triplicates and results are representative of 3 independent experiments.