DENND2B is a GEF for Rab13. (A and B) GST-Rabs, bound to glutathione beads, were incubated with HEK-293T cell lysates expressing the Flag-DENND2B DENN domain (A) or Flag–connecdenn 1 (CD1) DENN domain (B) in the presence of 5 mM EDTA. Ponceau S staining reveals the level of GST-Rabs, whereas specifically bound DENN domains were detected by blotting. Starting material (SM) equals 10% of the lysate used per condition. (C) Lysates from HEK-293T cells coexpressing Flag-DENND2B full length (FL) and GFP-Rabs were immunoprecipitated with the anti-GFP antibody in the presence of 5 mM EDTA, and the indicated proteins were detected by blotting. (D) GST-Rab13 Q67L, T22N, or GST alone, bound to glutathione beads, was incubated with HEK-293T cell lysates expressing Flag-DENND2B full length and analyzed as in A. (E) GST-RBD or GST alone, bound to glutathione beads, were incubated with HEK-293T cell lysates expressing GFP-Rab13 Q67L or T22N and analyzed as in A. (F–I) GST-RBD, bound to glutathione beads, was incubated with HEK-293T cell lysates expressing full-length Flag-DENND2B or DENN domain (F) or full-length Flag–connecdenn 3 (CD3) or DENN domain (H) and analyzed as in A. (G) Quantification of F (mean ± SEM in which n = 7 for mock and DENN domain and n = 6 for full length pooled from four independent experiments; one-way analysis of variance [ANOVA] with Dunnett’s post-test; *, P < 0.05; **, P < 0.01). (I) quantification of H (mean ± SEM in which n = 6 for mock and DENN domain and n = 5 for full length pooled from three independent experiments; statistical analysis used as in G).