The Vps9 domain of RabGEF1 is critical for GEF activity and the correction of −/− phenotypes. (A) Release of ³H-GDP (%) from Rab5 after a 30 minute incubation of ³H-GDP–loaded Rab5 with the indicated GST fusion proteins, as an assessment of GEF activity in vitro. Data shown are the mean + SEM from 3 separate experiments. ++ indicates P < .01; +++, P < .001 versus WT. (B) Percentage FcϵRI internalization (mean + SEM), determined as described in Figure 1C, from 5 (empty and WT) or 3 (D313A) separate batches of BMCMCs infected with the indicated lentiviral vectors. (C-D) IgE + Ag–induced (C) degranulation or (D) IL-6 production, determined as described in Figure 1D-E (mean + SEM of 9 separate determinations from 3 batches of BMCMCs). (E) IL-6 production by RabGEF1 +/+ or −/− BMCMCs sensitized for 16 hours in DMEM + 10% FCS containing 2 μg/mL IgE and stimulated for 6 hours in wells containing free or immobilized DNP at the indicated starting concentrations. (B-D) + indicates P < .05; ++, P < .01; +++, P < .001 versus corresponding +/+ “empty” values; * indicates P < .05; **, P < .01; ***, P < .001 versus corresponding −/− “empty” values. (E) # indicates P < .05; ##, P < .01 vs corresponding +/+ values. P < .01 vs corresponding +/+ values.