Ikaros expression and function is essential for μHC expression. (A) Immunoblot analysis for Pten and Ikaros expression in Pten-deficient cells transduced with an EV or with a Pten expression vector. Immunoblotting for actin served as loading control. (B) Immunoblot analysis of Ikaros and FoxO1 expression in Pten-deficient cells transduced with the indicated expression vectors. Immunoblotting for actin served as loading control. (C) Immunoblot analysis of FoxO1, Ikaros, and Pten expression in freshly isolated CD19⁺ BM cells from Pten-sufficient and Pten-deficient mice. Immunoblotting for actin served as loading control. (D) Flow cytometric analysis of Pten-deficient cells transduced with expression vectors encoding FoxO1-A3 and dominant-negative Ikaros (IK6). Single- (R1 and R2) and double-positive cells (R3) were gated and are displayed as histograms (right) showing µHC expression. Numbers indicate the percentages of cells expressing μHC. Data are representative of at least two to three independent experiments.