The coiled coil domain of RabGEF1 mediates Rabaptin-5 binding, but is not required for the correction of FcϵRI aggregation-induced −/− phenotypes. (A) RabGEF1 +/+ and −/− BMCMCs infected with the indicated constructs were subjected to immunoprecipitation with α-RabGEF1 (Ac-KSER) Abs (left panels) or α-HA Abs (right panels) and Western blot analysis with α–Rabaptin-5, α-HA, or α-RabGEF1 Abs, as indicated. (B) Pull-down assays on RabGEF1−/− BMCMC lysates with WT or CT2 RabGEF1 GST fusion proteins were analyzed by Western blot using α-Rabaptin-5, α-GST, and α-GAPDH Abs, as indicated. Results in panels A-B are representative of those obtained in 3 or more separate experiments. (C) Percentage FcϵRI internalization (mean + SEM), determined as described in Figure 1C, from 5 (empty and WT) or 3 (CT1 and CT2) 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.