Mitf expression rescues BMMC maturation defect because of p85α deficiency. (A) WT and p85α^−/− MCp cells were transduced with a retroviral vector expressing the Mitf cDNA. EGFP-positive cells were sorted to homogeneity and grown for additional weeks. Cells were harvested and subjected to Western blot analysis using an anti-Mitf or KIT antibody. The arrow in the top panel indicates the level of Mitf expression in various genotypes. The middle panel indicates the level of KIT expression in the various genotypes, and the lower bottom panel shows the level of GAPDH expression in each lane. n = 2. (B) Cells generated in panel A were harvested and subjected to flow cytometric analysis using antibodies against KIT and IgE receptor. The percentage of KIT and IgE receptor positive cells are indicated. Two representative experiments are shown. n = 3. (C-D) Reconstituting the expression of p85α in p85α-deficient MCps restored Mitf and KIT expression. MCps from WT or p85α-deficient mice were transduced with either the full-length form of p85α or the empty vector. Transduced cells were sorted on the basis of EGFP expression and cultured for 2 weeks. After 2 weeks, cells were harvested and subjected to Western blot analysis using an anti-KIT, anti-Mitf, or anti-GAPDH antibodies (C) or stained with antibodies against KIT or the IgE receptor and analyzed by flow cytometry (D). Percentages in each panel correspond to the level of KIT and IgE receptor levels in indicated genotypes. One of several independent experiments is shown.