VEGF-dependent phosphorylation of DEP-1 on Y1311 and Y1320 mediate Src activation in endothelial cells. (A) BAECS were transfected with empty vector (pmT2), WT DEP-1, and the indicated mutants, serum-starved, and then stimulated with VEGF (50 ng/mL) for 7 minutes. Src dephosphorylation on Y529 was detected using a phospho-specific antibody recognizing phosphorylated Y529. Densitometry ratios of p^Y529Src/Src levels were quantified using Quantity One software from Bio-Rad. As an indication of VEGF-dependent signaling, the activation of ERK1/2 was monitored by detecting phosphorylated T202/Y204. Equivalent signaling is observed in cells expressing WT DEP-1 and mutants. (B) BAECs were treated as in panel A. The phosphorylation of VE-cadherin was investigated after the immunoprecipitation of VE-cadherin and its immunodetection with the PY99 antibody. (C) HUVECs were serum-starved and stimulated with VEGF for the indicated times. Phosphorylation of endogenous DEP-1 is detected in total cell lysates (TCL) using phospho-specific antibodies detecting pY1311 and pY1320. The phosphorylation/activation status of Src on Y418 is determined by immunoblotting total cell lysates with the p^Y418Src antibody. Results suggest that VEGF-induced DEP-1 tyrosine phosphorylation is concomitant with Src activation. (D) The VEGF-mediated tyrosine phosphorylation of DEP-1 on Y1320 is enhanced in BAECs overexpressing WT DEP-1, but abrogated in cells expressing the Y1311F/Y1320F mutant (YY/FF). These results are representative of at least 3 independent experiments.