Deficiency of p85α in SHIP^−/− bone marrow cells rescues BMMC maturation in vitro. (A) Flow cytometric analysis demonstrating the expression of KIT and IgE receptor double-positive cells at the indicated times in BMMCs derived from the indicated genotypes. BM cells were harvested from WT, SHIP^−/−, p85α^−/−, and SHIP^−/−:p85α^−/− mice, and BMMCs were derived in vitro. Cells were harvested and stained with antibodies that recognize KIT and IgE receptor after the indicated weeks followed by flow cytometry. Numbers in the top right quadrant of each dot blot indicate the percentage of BMMCs that are double positive for KIT and IgE receptor expression during different times of culture. n = 5. (B) Hyperactivation of AKT in SHIP^−/− BMMCs is reduced in the setting of p85α deficiency. BMMCs derived from the indicated genotypes were starved in the absence of growth factors and stimulated for the indicated times. Cell lysates were subjected to Western blot analysis using antibodies that recognize the activated version of the indicated signaling proteins. Similar findings were observed in additional 1 to 3 independent experiments. (C) Loss of SHIP expression in BMMCs results in enhanced expression of Mitf. BMMCs derived from WT, SHIP^−/−, p85α^−/−, and SHIP^−/−:p85α^−/− mice were lysed and 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. n = 3.