Absence of both CD19 and BCAP impairs in vitro B-cell differentiation and BCR-mediated Akt activation. (A) Sorted CD43⁺B220⁺IgM⁻ cells from bone marrows (5 × 10⁵) were cultured in the presence of 20 ng/mL murine recombinant IL-7 for 4 days. The numbers of recovered cells were counted by trypan blue exclusion. Data represent the means of recovered cell numbers (± SD) of triplicate cultures. (B) Bone marrow B cells were prepared from wild-type, BCAP^−/−, CD19^−/−, or CD19^−/−BCAP^−/− mice and cultured for 4 days in the presence of IL-7. The cells were washed with medium twice and cultured in the absence (− IL-7) or presence (+ IL-7) of IL-7 for an additional 2 days. Approximately 50% of cells were recovered from each genotype after 2 days in the absence of IL-7. The recovered cells were stained with anti-IgM and anti-IgD Abs and subjected to flow cytometric analysis. The percentage of IgM⁺IgD⁺ and IgM⁺IgD⁻ cells is indicated in each panel. (C) Bone marrow B cells were cultured for 4 days in the presence of IL-7. The recovered cells were stimulated with 30 μg/mL anti-Igβ mAb for the indicated periods of time. Whole-cell lysates from stimulated cells were subjected to Western blotting with anti–phospho-serine 473, anti–phospho-threonine 308 Akt, or anti-Akt1 antibodies. (D) AA4.1⁺B220⁺ B cells were sorted from the splenocytes of wild-type or BCAP^−/−CD19^−/− mice. The cells were stimulated with anti-Igβ mAb at a concentration of 30 μg/mL for 3 minutes. Total-cell lysates were prepared and subjected to Western blotting analysis.