VEGF₁₆₅ specifically inhibits Sema3A binding to endothelial, stromal, and neuronal cells. (A) Concentration dependency of Sema3A/Fc (○) binding to endothelial cells. Control B7-1/Fc (16 μg/mL, □). (B) Endothelial cells were incubated (4°C, 1 hour) with Sema3A/Fc (2 μg/mL) plus VEGF₁₆₅ (0-100 ng/mL), VEGF₁₂₁ (100 ng/mL), VEGF-B₁₆₇ (100 ng/mL), VEGF-C (100 ng/mL), or FGF-2 (100 ng/mL). (C) Endothelial cells were preincubated (25°C, 1 hour) with or without VEGF₁₆₅ (12.5 ng/mL), VEGF₁₂₁ (100 ng/mL), or FGF-2 (100 ng/mL). Sema3A/Fc (2 μg/mL) was added and cells were further incubated at 4°C for 60 minutes. (D) Human stromal HS-5 and (E) rat pheochromocytoma PC12 cells were preincubated (25°C, 1 hour) with VEGF₁₆₅ (0-250 ng/mL) or VEGF₁₂₁ (250 ng/mL). Sema3A/Fc (2 μg/mL) was added and cells were further incubated at 4°C for 60 minutes. Sema3A/Fc binding was detected by anti-Fc mAb. Shaded graphs reflect control staining.