In situ ligand-binding assay were performed as described previously (60). Briefly, COS-7 cells transiently transfected to express either full-length Sema4C or KG100-101 → DT mutated Sema4C were incubated for 1 hour at 37°C with a recombinant soluble PlexinB2 extracellular domain fused to alkaline phosphatase (AP). The latter had been harvested from the conditioned medium of COS-7 cells transiently transfected with a cDNA construct encoding PlexinB2 sequence comprised between amino acids 1 and 1190, fused to secreted placental AP. After five washes, Sema4C-expressing cells were fixed, heated for 10 min at 65°C to inactivate endogenous phosphatases, and incubated with nitro blue tetrazolium-5-bromo-4-chloro-3-indolyl-phosphate AP substrate (Promega) to reveal bound PlexinB2.

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