Δ21-161 EMCN does not interact with VEGFR2 or rescue VEGFR2 internalization. (A,B) Membrane proteins of HRECs lacking endogenous hEMCN and overexpressing the different Myc-tagged mEMCN constructs were extracted and subjected to co-immunoprecipitation (co-IP) using anti-Myc antibody, and the protein levels of VEGFR2 and the different mEMCN mutant proteins were examined by western blot. The FL EMCN, Δ21-81, and Δ21-121 all co-IP’ed with VEGFR2 whereas Δ 21-161 did not. (C,D) To assess VEGFR2 internalization, cell surface proteins were labeled using NHS-SS-biotin and isolated, and protein levels of VEGFR2 and CD31 were analyzed by western blot. The FL EMCN, Δ21-81, and Δ21-121 all rescued VEGFR2 internalization; Δ21-161 did not. All data = mean ± SEM, ns, not significant, * p < 0.05, **** p < 0.0001 by 2-tail unpaired t-test, n = 4.