Either NRP1, a NRP1 mutant deleted FV/FVIII and MAM domains (NRP1Δ 276–797), or a partial deletion of CUB domain (NRP1Δ 18–253) was expressed in COS-7 cells. Sema3A and its deletion mutants were fused with human placental alkaline phosphatase (AP). The cells were incubated with AP-Sema3A (300pM), Sema-Ig-AP (4nM), or AP-basic rich region (1nM) for 60 min. Bound AP fusion proteins were visualized. NRP1Δ 276–797 bound the Sema-Ig but not the basic rich region, whereas NRP1Δ 18–253 bound the basic rich region, suggesting the CUB domain is the binding site for the Sema domain of Sema3A. Scale bar, 100 mm.