Sbf promotes GTP exchange on Rab35 GTPase. (A) Purified MBP:DENN pulls down His:Rab35, but not His:Rab11. (B) Preincubation of His:Rab35 with GMP-PNP decreases Rab35 pull-down by MBP:DENN. (C) Quantification of the normalized protein levels of His:Rab35, His:Rab11, and GMP PNP-His:Rab35 in pull-down assays in A and B. The intensity was normalized to MBP fusion protein band intensity. (D) GTP-exchange rates of Rab35 and Rab11 in the presence or absence of MBP:DENN. Adding unlabeled GMP-PNP (arrow) competes with the labeled GTP assay. Fluorescence was measured and normalized to the fluorescence at time 0 s. (E) Quantification of normalized fluorescence intensity changes in D (n = 5 trials). Statistical significance was calculated using a Student’s t test (C) and Wilcoxon test (E). *, P < 0.05; **, P < 0.005.