Effect of B cell-targeted expression of PKCβII on B cell populations in splenic tissue from Eµ-PKCβIItg and wt mice. Single cell suspensions prepared from spleens isolated from Eµ-PKCβIItg and wt mice were stained with a cocktail of antibodies containing B220, IgM, IgD, CD43, CD21 and CD24 markers, and then analysed by flow cytometry. Quantitative comparisons were then made of the percentage B220+ cells within the following gates (A) IgD+ IgMdim, (B) IgD+ IgM+, and (C) IgD+ IgM+ CD43− CD21+ CD24+ (MZ-like B cell) for wt and Eµ-PKCβIItg mice as defined by the strategy illustrated in Supplementary Figure 2. Black dots in these graphs refer to wt and homozygous Eµ-PKCβIItg progeny derived from mating heterozygous Eµ-PKCβIItg mice. Red dots refer to wt mice with similar genetic backgrounds (C57BL/6) that are alike in terms of age to the transgenic mice. (D) upper panels H&E staining of splenic tissue from wt and Eµ-PKCβIItg mice. Lower panels anti-IgM staining of spleen sections from wt and Eµ-PKCβIItg mice. These images are representative of n = 2 experiments using splenic tissue from different mice that had been aged in excess of 12 months. Inset arrows indicate MZ. These histogram images have been published in the PhD thesis of AAA43. (E) BCR-induced Ca2+ flux in isolated splenic B cells from heterozygous and homozygous Eµ-PKCβIItg mice. Total flux was calculated as area under the curve is reported in arbitrary units. Statistical analysis for parts (A) (*P = 0.012), (B) (*P = 0.016), (C) (**P = 0.0052) and (E) (*P = 0.024) was performed using a Mann–Whitney U test.