Overexpression of GEM-Cbl-b dramatically inhibits the FcϵRI-induced tyrosine phosphorylation of Gab2 and stimulates the degradation of Gab2. (A) Control cells and cells overexpressing GEM-Cbl-b and GEM-Cbl-b (C373A) were primed with anti-DNP IgE and stimulated with 30 ng/mL antigen DNP-BSA for the indicated times. Cell lysates (10⁷) were solubilized in the denature buffer and immunoprecipitated with anti-Gab2 antibody. The immunoprecipitates and detergent-soluble cell lysates were separated by SDS-PAGE and analyzed by the immunoblotting with anti-pTyr mAb and anti-Gab2 antibody. The numbers at the bottom of the figures were the normalized densitometric analysis of Gab2 phosphorylation (Gab2 tyrosine phosphorylation/Gab2 protein). The levels of Gab2 expression were 20% (GEM-Cbl-b overexpressing cells) and 90% (GEM-Cbl-b [C373A] overexpressing cells) compared with the control cells. (B) Cells stably expressing Flag-Ub were primed with anti-DNP IgE and stimulated with 1 μg/mL antigen DNP-BSA for the indicated times. Either nonstimulated or antigen-stimulated cell lysates (3 × 10⁶) were solubilized in the denature buffer and immunoprecipitated with anti-Gab2 antibody. The immunoprecipitates were separated by SDS-PAGE and analyzed by the immunoblotting with anti-Flag mAb and anti-Gab2 antibody. In the immunoblotting with anti-Gab2 antibody, the membrane was exposed for 1 minute (short exposure) and 20 minutes (long exposure). Ubiquitination of Gab2 (Gab2-Ub) was indicated. Similar results were obtained when the other cloned lines were examined. The levels of Gab2 expression were 60% (1 minute) and 50% (3 or 10 minutes after the antigen stimulation) compared with the unstimulated cells. The results were representative of 4 experiments.