Loss of p85α in MCps results in altered expression of key transcription factors associated with mast cell maturation. (A) BM cells from WT and p85α^−/− mice were cultured for 3 weeks, after which cells were stained with Giemsa (bottom) or antibodies that recognize Gr-1 and Mac-1 followed by flow cytometry (top). Shown is a dot blot profile from 1 of 4 independent experiments. Percentage of Gr-1 and Mac-1 double-positive cells are indicated in the top right quadrant. (B) Differential expression of Mitf and Gata-2 in WT and p85α-deficient cells. Cells described in panel A were lysed, and equal amounts of protein lysate were subjected to Western blot analysis using an anti-Mitf, anti-Gata-2, anti-PU.1, and anti–β-actin antibody as indicated. Shown is a representative Western blot from 2-3 independent experiments. (C) Overexpression of Gata-2 in WT BMMCs mimics the p85α^−/− differentiation phenotype. WT BMMCs transduced with vector, Mitf, and Gata-2 alone or in combination were sorted to homogeneity and cultured for 3 to 4 weeks. Maturation of BMMCs and Gr-1/Mac-1 myeloid cells was analyzed by staining the cells with antibodies that recognize KIT and IgE receptor as well as the presence of Gr-1 and Mac-1 by flow cytometry. Shown are dot blots from 1 representative experiment performed 2-3 independent times.