FIGURE 6:

The Arl4A-Robo1 interaction induces Cdc42 activation via reducing the association between Robo1 and srGAP1. HEK293T cells were transiently transfected with an (A) empty vector, srGAP1, Flag-Robo1-WT, Flag-Robo1-A1, and Arl4A or (B) srGAP1, Flag-Robo1-WT, and Arl4A. The interaction was verified by in vivo coimmunoprecipitation with anti-Flag M2 magnetic beads. To confirm the initial expression level, 2.5% of the total cell lysate (input) was loaded. Equal amounts of cell lysates were used in each experiment as shown by Coomassie Blue staining. The heavy chain was used to ensure equal amounts of M2 magnetic beads were loaded in each group. SDS–PAGE analysis was used to verify srGAP1, Robo1, and Arl4A expression and total cell lysates. Histograms: Co-IP assay data were quantified based on three biological replicates. The solid bars represent the mean ± SD. *, P < 0.01; **, P < 0.005; ***, P < 0.001 (A: two-tailed Student's t test; B: one-way ANOVA with Dunnett's post hoc multiple comparison test).