SH3BP1 forms a complex with PlexinD1 via its BAR domain, and Sema3E binding to PlexinD1 activates SH3BP1 by releasing it from the PlexinD1 complex. (A) Schematic illustration of the SH3BP1 constructs used in this study. The full-length SH3BP1 construct contains BAR and RhoGAP domains and SH3 motifs on the C-terminal end. In the SH3BP1ΔRhoGAP construct, the GAP domain was deleted. The SH3BP1ΔBAR construct lacked the BAR domain, and in the SH3BP1ΔSH3 construct the SH3 motif was deleted. (B) SH3BP1 is associated with PlexinD1 through the SH3BP1 BAR domain. HEK293T cells were transfected with PlexinD1-GFP and vectors expressing SH3BP1 (SH3BP1-HA or deletion constructs SH3BP1ΔBAR-HA, SH3BP1ΔRhoGAP-HA, and SH3BP1ΔSH3). Cells were lysed and immunoprecipitated with a GFP antibody and immunoblotted with an HA antibody (right). PlexinD1 and SH3BP1 expression levels in the cell lysates were determined by Western blotting with GFP and HA antibodies (left). (C) SH3BP1 dissociated from PlexinD1 upon Sema3E treatment. HEK293T cells transfected with PlexinD1-GFP and SH3BP1-HA were treated with Sema3E for 0, 10, and 20 min, then lysed and immunoprecipitated with a GFP antibody and immunoblotted with an HA antibody. The dissociation of SH3BP1 from PlexinD1 was observed 20 min after Sema3E treatment, as indicated on the right, while the protein expression levels are shown on the left.