(A and B) Representative sensorgrams of 3 experiments showing binding of cANGPTL4 to immobilized NRP1 and NRP2, using SPR binding analysis. KA and KD constants are shown. The complete kinetics analysis is listed in Supplemental Table 3. cANGPTL4 concentrations are 6.25 nM, 12.5 nM, 25 nM, and 50 nM for binding of ANGPTL4 to NRP1 (A) and 12.5 nM, 25 nM, and 50 nM for binding of ANGPTL4 to NRP2 (B). (C) Immunoprecipitation of 5 μg/mL rhcANGPTL4 with endogenous NRP1 and NRP2 in HUVECs. Levels of VEGF, cANGPTL4, NRP1, NRP2 and actin were determined in gels run in parallel. (D) Rho assay upon transfection of scrambled siRNA, NRP1 siRNA, or NRP2 siRNA and treatment with (5 g/mL) rhANGPTL4, rhcANGPTL4, or rhnANGPTL4 or none (control) in HUVECs. One-way ANOVA (D). ***P < 0.001. Experiments were repeated at least 3 times.