Sema3A and VEGF require Nrp-1 expression to induce vascular permeability. (A) To determine knockdown of Nrp-1 expression in inducible, endothelial-specific knockouts, mice were injected with 20 mg/mL tamoxifen (Tam) for 2 weeks to excise the floxed exon. Heart lysates from EC-SCL-CreERT–positive or –negative Nrp-1fl/fl mice were immunoblotted with antibodies against Nrp-1 or ERK2 as a loading control. (B) Cryosections (5 μm) from Tam-treated EC-SCL-CreERT–positive or –negative Nrp-1fl/fl hearts were stained with antibodies against Nrp-1 (red) and the endothelial markers (EC, green). Confocal images were taken at 400× magnification (scale bar represents 50 μm). (C) Knockdown of Nrp-1 expression disrupts Sema3A- and VEGF-mediated VP. Mice treated with Tam or vehicle were injected subcutaneously with Sema3A, VEGF, or PBS. Dye extravasation was quantitated (n = 8, *P < .05). (D) A function-blocking monoclonal antibody against Nrp-1 inhibits Sema3A- but not VEGF-induced VP. Balb/c mice were injected with an anti–Nrp-1 or IgG (50 μg/mL intravenously) 30 minutes prior to injection of VEGF, Sema3A, or PBS (n = 9, *P < .05). Error bars represent SEM.