FIGURE 5.

Affinity interaction between IR800-endorepellin and the VEGFR1/2 and attenuation of VEGFA-evoked activation of VEGFR2 in HUVECs. A and B, ligand-binding assays using IR800-endorepellin as a soluble ligand and VEGFR1 or 2 as immobilized substrates. C and D, displacement assay using constant molar amounts of IR800-endorepellin (10 nm) and increasing molar amounts of unlabeled endorepellin as indicated. Values represent the mean ± S.E. of three independent experiments run in triplicates. E, pulldown of IR800-endorepellin utilizing VEGFR1-Fc immobilized to protein A-Sepharose magnetic beads. Note that IR800-endorepellin is specifically bound to the magnetic beads linked to the VEGFR1-Fc (+). The left panel is visualized with the Odyssey infrared image analysis system. Note that the molecular mass markers emit at ∼680 nm and are visualized in red. The right panel shows the same gel after staining with Coomassie Blue to detect the VEGFR1-Fc (arrow). F, similar experiment as described in E. Note that also in this case, IR800-endorepellin binds specifically to VEGFR2-Fc (+) (left panel) and co-localizes with the presence of VEGFR2-Fc (arrow in right panel). Notice also that VEGFR2-Fc migrates slower than VEGFR1-Fc due to its larger size (i.e. an extra Ig repeat). G, representative immunoblot of total proteins from HUVECs treated with VEGFA, endorepellin, or the VEGFR2 kinase inhibitor SU5416 as indicated. Notice that endorepellin treatment inhibits the phosphorylation of VEGFR2 at Tyr-1175 analogous to SU5416. Total VEGFR2 and GAPDH levels provide loading controls. H, representative co-immunoprecipitation studies using anti-VEGFR2 antibodies. Note that the α2 integrin subunit can be co-immunoprecipitated only in the presence of endorepellin. Induction of Tyr-1175 phosphorylation by VEGFA and blockage by endorepellin indicate that both ligands were biologically active. The experiments were repeated three times with comparable results.