VEGF eludes sVEGFR-1 and access endothelial cells in the presence of MMP-7. (A) Phosphorylation of VEGFR-2 after treatment of HUVECs with VEGF + sVEGFR-1 + MMP-7. sVEGFR-1 (91.4 ng/mL) was incubated with MMP-7 (28.6 ng/mL) for 48 hours, with VEGF (20 ng/mL) overnight, and with anti-VEGF antibody for 30 minutes. The sample solutions were added to HUVECs for 5 minutes, and the treated cells were analyzed by Western blotting with antibody against phospho-VEGFR-2 (Tyr1054/1059). The total amount of VEGFR-2 was also measured. (B) Coimmunoprecipitation assay of exogenous VEGF and endothelial sVEGFR-1. Recombinant VEGF₁₆₅ was added to the supernatant of HUVECs for 24 hours, and the supernatant was incubated with MMP-7 for 24 hours and then immunoprecipitated with anti-VEGF antibody. The precipitated proteins and unbound supernatant fluids were analyzed for sVEGFR-1 and VEGF by Western blotting.