p50α and p55α isoforms of p85 are important for mast cell maturation. (A) Schematic of regulatory subunits p85α, p55α, and p50α. (B) Deletion of all regulatory subunits of p85α. To delete all regulatory subunits of p85α (p85α, p55α, and p50α) in BM cells, p85α^fl/fl/ Mx-Cre⁻ mice (WT), and p85α^fl/fl/Mx-Cre⁺ mice (p85α^fl/fl−/−) were injected intraperitoneally with poly I:C (300 μg) 3 times on alternate days, and BM was harvested 3 weeks after the final injection. BM from WT and p85α^fl/fl−/− mice was cultured, and equal amounts of protein lysate were subjected to Western blot analysis using a pan–anti-p85 antibody (this antibody recognizes all regulatory subunits of class I_A PI3K) to confirm the deletion of PI3K subunits. (C) Deficiency of p50α and p55α further impairs the maturation of p85α^−/− BMMCs. BM cells from WT, p85α^−/−, and p85α^fl/fl−/− mice were cultured, and at indicated time points mast cell maturation was analyzed by staining the cells with antibodies that recognize KIT and IgE receptor by flow cytometric analysis. Shown is a dot blot profile of 1 of 3 independent experiments. (D) Expression of p50α in p85α^−/− BMMCs. MCps from WT and p85α^−/− mice were transduced with vector, HA-tagged full-length p85α, or p50α and sorted to homogeneity. Expression of p85α and p50α in sorted cells was analyzed by Western blotting using anti-HA and β-actin antibodies. (E-F) Restoration of p50α corrects the defective maturation of p85α-deficient BMMCs. BMMCs in panel D were collected at indicated time points. Mast cell differentiation was analyzed by staining the cells with antibodies that recognize KIT and IgE receptor followed by flow cytometric analysis. Shown is a representative dot blot profile (E) and quantitative data (F) from 5 independent experiments. *P < .05, WT versus p85α^−/−vector.