DMP1 affects VEGFR-2, not VEGFR-1, phosphorylation, induces Src inactivation, and inhibits VEGF-mediated VE-cadherin down-regulation and phosphorylation. (A) Western blot analysis with antibodies to VEGFR-2 and VEGFR-1 and their phosphorylated forms using total lysates from HUVECs treated with DMP1 for 24 hours. (B) Western blot analysis with antibodies to P-VEGFR-2 and VE-cadherin using total lysates from HUVECs transfected for 48 hours with VE-cadherin or nontargeting siRNAs and treated with DMP1 (50 nmol/L) for the last 24 hours of transfection. (C) Western blot analysis with antibodies to VEGFR-2 and P-VEGFR-2 using total lysates from HUVECs treated with DMP1 for 24 hours and then pulsed with VEGF (50 ng/mL) for a further 10 minutes. (D) Western blot analysis with specific antibodies to Src, P-Src Tyr416, and P-Src Tyr527 using total lysates from HUVECs treated with DMP1 and VEGF as in panel C. (E) Western blot analysis with antibodies to VE-cadherin and P-VE-cadherin Tyr658, Tyr731, and Tyr685 using total lysates from HUVECs treated with DMP1 and VEGF as in panel C. (F) Western blot analysis with an antibody to Csk using total lysates from sparse and confluent HUVECs treated with DMP1 for 24 hours. (G) Western blot analysis with an antibody to Csk using total lysates from HUVECs treated with DMP1 and VEGF as in panel C. All Western blots were evaluated by densitometric scanning (in italics below the lanes) and were performed 3 times with similar results. Equal protein loading was assessed by anti–β-actin or anti–Hsc 70 immunoblotting.