The ZnF domain of RabGEF1 mediates ubiquitination in vitro, but is not required for the correction of FcϵRI aggregation-induced −/− phenotypes. (A) In vitro ubiquitination assays with WT or mutant RabGEF1 GST fusion proteins were performed + Ub and UbcH5a (E2), and ± Ub-activating enzyme (E1), as indicated. Samples were analyzed by Western blot using α-Ub and α-RabGEF1 Abs. Arrows indicate autoubiquitinated RabGEF1. (B) In vitro Ub pull-down assays (left panel) with WT or mutant RabGEF1 GST fusion proteins were performed in the presence of poly-Ub chains (Ub_1-7, K48-linked) and analyzed by Western blot using α-Ub and α-GST Abs. Results in panels A and B are representative of those obtained in 4 or more separate experiments. Signals from 4 separate experiments were quantified by densitometric scanning and corrected for loading (B, right panel); data shown are mean + SEM. + indicates P < .05, +++, P < .001 versus WT. (C) Percentage FcϵRI internalization (mean + SEM), determined as described in Figure 1C, from 5 (empty and WT) or 3 (NT and ZnF) separate batches of BMCMCs infected with the indicated lentiviral vectors. (D-E) IgE + Ag–induced (D) degranulation or (E) IL-6 production, determined as described in Figure 1D-E (mean + SEM of 9 separate determinations from 3 batches of BMCMCs). (C-E) ++ indicates P < .01; +++, P < .001 versus corresponding +/+ “empty” values; * indicates P < .05; **, P < .01; ***, P < .001 versus corresponding −/− “empty” values.