IBtkγ S87/90A functions as super-repressor of Btk-dependent Ca²⁺ mobilization and NF-κB activation in response to BCR triggering. (A) Profile of Ca²⁺ mobilization in DeFew cells stably expressing the wild-type FLAG-IBtkγ- IRES-GFP or mutants. DeFew cells (1 × 10⁷) were loaded with the Ca²⁺ indicator Rhod-2 and stimulated with anti–human IgM (20 μg/mL). The maximum Ca²⁺ release was measured after the addition of calcium ionophore ionomycin (1μM). The GFP and Rhod-2 fluorescences were acquired by FACS during 180-second time periods and analyzed by FlowJo software (TreeStar Inc). For each experimental point, either cytogram of GFP-positive and GFP-negative subsets, and histogram showing the profile of Ca²⁺ mobilization in the GFP subsets of each cell line, are reported. Ca²⁺ mobilization is shown as the Rhod-2 fluorescence intensity compared with the baseline (calculated as 1). A representative experiment of 3 independent experiments is shown. (B) Cumulative profiles of Ca²⁺ mobilization on anti-IgM (top), or ionomycin (bottom) for the GFP-positive gated populations of the experiment described in A. (C) NF-κB activation in response to BCR triggering in DeFew cells expressing FLAG-IBtkγ or mutants. DeFew cells (6 × 10⁶) stably expressing the wild-type or mutant FLAG-IBtkγ-IRES-GFP were transfected with NF-κB-luc (15 μg) and renilla reporter plasmids (0.5 μg); 24 hours later, cells were treated for 18 hours with anti-IgM (20 μg/mL) or left untreated. The luciferase and renilla activities were measured in cells extracts at least in triplicate and luciferase activity was normalized as the ratio between the luciferase and renilla activity. Statistical significant difference between the empty vector and the vector encoding the wild-type or mutant IBtkγ was analyzed by Student t test. A representative experiment of 3 independent experiments is shown.