SH3BP1 was identified from the screen as a downstream component of Sema3E-PlexinD1 signaling, and further validated by siRNA-resistant SH3BP1 rescue and PlexinD1/SH3BP1 colocalization. (A) Automated image acquisition showed that SH3BP1 knockdown blocked Sema3E-induced collapse to similar extent as PlexinD1 siRNA. HUVECs treated with Sema3E underwent cell collapse that was inhibited by transfection with siRNA targeted specifically to PlexinD1 or SH3BP1. Bar, 100 µm. (B) Quantification of the Sema3E-induced cell collapse using the automated image analysis algorithm. ***, P < 0.0001. Error bars indicate SEM. (C) PlexinD1 and SH3BP1 colocalized at the leading edge of the cell (arrows). PlexinD1-GFP and SH3BP1-HA were coexpressed in HUVECs and stained with the corresponding antibodies. Bar, 10 µm. (D) Endogenous localization of PlexinD1 and SH3BP1 in HUVECs. PlexinD1 and SH3BP1 colocalized in the leading edge of the cells (arrows). The boxed regions are enlarged on the right. Bar, 10 µm. (E) Reintroducing SH3BP1 protein rescued SH3BP1 siRNA inhibition of Sema3E-induced collapse. HUVECs were transfected with SH3BP1 siRNA followed by transfection with control DNA or the siRNA-resistant SH3BP1 construct and treated with Sema3E or control ligand. Strong cell collapse was observed only in cells transfected with the rescue construct (arrows). Cell shape was visualized by DTAF labeling (green) and vector expression was detected by an HA antibody (red). Bar, 100 µm. (F) Quantification of the cell collapse demonstrated the ability of the siRNA-resistant SH3BP1 to fully rescue the Sema3E-induced collapse. **, P < 0.001. Error bars indicate SEM.