Ibtkγ^−/− spleen B cells show a sustained Btk-Y551 phosphorylation and intracellular Ca²⁺ signaling in response to BCR-engagement. (A) Percentage of the splenic B-cell subsets in 5-week-old mice. Spleen cell suspensions derived from 5-week-old mice were stained with the following antibodies: CD21-FITC, CD23-PE, AA4.1-APC, IgM-APC-Cy7, and analyzed by flow cytometry. Percentage for Ibtkγ^+/+ and Ibtkγ^−/− spleen cells for T1 (CD21^negCD23^negAA4.1^posIgM^pos), T2 (CD21^posCD23^posAA4.1^posIgM^pos), follicular mature (CD21^posCD23^posAA4.1^negIgM^pos), and marginal zone (MZ; CD21^highCD23^negAA4.1^negIgM^pos) type B cells are reported (n = 5). Statistical analysis was performed with Prism v.4.0 (GraphPad software) and included the unpaired 2-tailed t test. (B) Ibtkγ^−/− spleen B cells show a sustained Btk-Y551 phosphorylation on BCR engagement. Purified splenic B cells from either wild-type or Ibtkγ^−/− 5-week-old mice were stimulated with F(ab′)₂ fragments of anti–mouse IgM (10 μg/mL) for the indicated time and then analyzed by Western blotting with the indicated antibodies. The amounts of phosphorylated Btk are reported as relative optical density and evaluated according to the intensity of the phospho-Btk signal normalized to the corresponding Btk signal. A representative experiment of 3 independent experiments is shown. (C) Profile of Ca²⁺ mobilization in splenic B cells from wild-type or Ibtkγ^−/− mice. Purified spleen B cells (1 × 10⁷) from either wild-type or Ibtkγ^−/− 5-week-old mice were loaded with the Rhod-2 Ca²⁺ indicator and stimulated with F(ab′)₂ fragments of anti–mouse IgM (10 μg/mL). The maximum Ca²⁺ release was measured after the addition of calcium ionophore ionomycin (1μM). 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.