Syb2 interacts with Nrp1 and PlexA1. (A) Immunoprecipitations were performed on E15 brains using Syb2 and mouse control immunoglobulin antibodies. Coimmunoprecipitated proteins were identified by Western blotting. N-cadh, N-cadherin. (B) Syb2 and control immunoprecipitations were performed on E15 brains and adult brain. (C) Representation of GFP-Syb2 and Syb2-SEP constructs and the TeNT cleavage site. mb, membrane; closed box, GFP-Syb2 not cleaved; open box, GFP-Syb2 cleaved by TeNT; closed circle, Syb2-SEP not cleaved; open circle, Syb2-SEP cleaved by TeNT. (D) HEK-293T cells were transfected with GFP-Syb2; Syb2-SEP, Nrp1-mRFP, PlexA1-FLAG, and mutant; or wild-type TeNT plasmids. Lysates were immunoprecipitated with GFP, RFP, FLAG, and control immunoglobulin antibodies. After elution, Syb2-GFP/SEP, Nrp1-mRFP, and PlexA1-FLAG were identified by Western blotting through their respective tags. Boxes with broken lines, interacting domains. Black lines indicate that intervening lanes have been spliced out.