Functional siRNA screen for Rho family GEFs and GAPs. (A) Basic screen design: (1) ephrinB⁺ SKN-H2B-RFP cells were seeded into 96-well imaging plates and library of siRNA oligos were added. (2) Cells were incubated for 72 h and (3) co-cultured for 80 min with HeLa EphB2ΔC-GFP/Flag cells prestained with CellTracker green. (4) Cells were then fixed without permeabilization and immunostained against Flag. (5) Cells were imaged, and (6) semiautomated data analysis was performed using CellProfiler. On average, 150 responder cells per well were analyzed. (B and C) Heatmap for mean z-score values of the two independent runs showing all four siRNA oligos (A–D) per gene for all GEFs or GAPs, respectively. Specific GEF or GAP activity for each GTPase subfamily is indicated, taken from previous studies (Tcherkezian and Lamarche-Vane, 2007; Jaiswal et al., 2013; Cook et al., 2014) and independent literature search (+, active toward at least one member of subfamily; −, negative for all members of subfamily; /, unknown specificity). Genes are ranked from mean z-score of all four siRNA oligos. Values shown by intensity profile. (D) Quantification showing the requirement of Tiam2 for EphB2ΔC trans-endocytosis into ephrinB⁺ SKN cells. Experiment design and analysis as described in A. Results shown as mean ± SE, (n = 5 independent experiments, 44–449 responder cells per condition per experiment). ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001, repeated measures one-way ANOVA with Dunnett’s post hoc test. (E) Example images of scramble siRNA controls and top-ranked hits. Total EphB2ΔC is shown in green, surface EphB2ΔC in red, and SKN-H2B in blue. Bars: 20 µm; (inset) 2.5 µm.