Expression of activated PI3K (p110CAAX) accelerates the rate of WT BMMC maturation by inducing the expression of Mitf. WT MCps were transduced with either an empty vector control or a vector expressing an activated version of PI3K (p110CAAX). Transduced cells were selected in puromycin for 2-3 weeks and analyzed for maturation. (A) Cells were subjected to flow cytometric analysis to assess the coexpression of KIT and IgE receptor double-positive cells after culture. Numbers in the top right quadrant indicate the percentage of cells that are double positive for KIT and IgE receptor expression. Shown are representative dot blots from 1 of 4 independent experiments. (B) Lysates derived from cells in panel A were subjected to Western blot analysis using antibodies against phospho-AKT and total AKT. The level of activated AKT is indicated by an arrow. (C) Lysates derived from cells in panels A and D were subjected to Western blot analysis using an anti-Mitf antibody. Arrows indicate the level of expression of Mitf and β-actin (loading control) in each lane. (D) Expression of activated AKT in p85α^−/− BMMCs rescues mast cell differentiation. MCps from WT or p85α^−/− mice were transduced with retrovirus expressing either an empty vector or an activated version of AKT with an HA tag. Transduced cells were cultured for another 2 to 3 weeks, and EGFP-positive cells were sorted and analyzed for the expression of KIT and IgE receptor by flow cytometry. Percentage of KIT and IgE receptor double-positive cells is indicated in the top right quadrant, n = 3. (E) Cells generated in panel D were subjected to Western blot analysis using an anti-HA, anti-Mitf, anti-KIT, and anti–β-actin antibody. The level of expression of KIT, Mitf, HA-tagged AKT, and β-actin is indicated. Cells generated in panel D also were analyzed for AKT activation (bottom 2 panels).