Expansion of Btk^−/−PLCγ2^−/− pre–B cells in the presence of IL-7. (A) Proliferation of pre–B cells in IL-7 culture. CD19⁺ B cells from bone marrow of WT, Btk^−/−, PLCγ2^−/− and Btk^−/−PLCγ2^−/− (DKO) mice were cultured in the presence of various doses of IL-7, and the number of cells was counted after 4 days of treatment. The proliferative capacity was defined as the increase in cell number relative to the input number of cells at day 0. (B) FACS analysis of cultured pre–B cells. Cells in a 9-day culture were analyzed for the expression of B220, Igκ/λ, and pre-BCR. Numbers indicate percentage of cells in the live cell gate (top panels) and percentage of B220⁺IgL⁻ cells (bottom panels). (C) Western blot analysis of pre-BCR–induced ERK activation. Cultured pre–B cells were stimulated with 10 μg/mL anti-Igμ F(ab′)₂ antibody for various periods of time, and whole-cell lysates were probed with anti–phospho-ERK antibody. Blots were reprobed with anti-ERK2 antibody to determine protein loading. Results shown are representative of 4 and 3 independent experiments for panel A and panels B-C, respectively.