Mice with germline SHIP deficiency have an expanded CD25⁺ T_reg compartment in peripheral lymphoid organs and higher percentages of thymic CD4⁺FoxP3⁺ T_regs than WT counterparts. Spleens, mesenteric LN, and thymuses of SHIP^−/−, SHIP^ΔIP/ΔIP, and their respective WT controls were harvested from 6- to 9-week-old mice and processed into single-cell suspensions. In spleens and LN, CD3⁺CD4⁺CD25⁺ T_regs were quantified using a CD3, CD4, CD25, and FoxP3 multicolor stain. In thymuses, CD4⁺CD8⁻CD25⁻FoxP3⁺ T cells and CD4⁺CD8⁻CD25⁺FoxP3⁺ T_regs were quantified using a CD4, CD8, CD25, and FoxP3 multicolor stain. (A) Representative CD4 versus CD25 staining after gating on CD3⁺ T cells for spleen and LN of SHIP^ΔIP/ΔIP and WT littermates. (B) Percentage frequency of CD4⁺CD25⁺FoxP3⁺ T_regs after gating on CD3⁺ T cells, and total absolute CD3⁺CD4⁺CD25⁺FoxP3⁺ T_reg numbers, respectively, in the spleen and LN of the indicated genotype. For SHIP^ΔIP/ΔIP mice: n = 12 and littermate control: n = 10. For SHIP^−/− mice: n = 6 and littermate control: n = 6. (C) Representative FACS analysis of FoxP3 expression CD3⁺CD4⁺CD25⁺ T_regs in the spleen of the indicated genotype. Western blot analysis of SHIP, FoxP3, and β-actin expression in lysates prepared from sorted CD3⁺CD4⁺CD25⁺ T_regs of the indicated genotype. AFU values (“Western blotting”) for FoxP3 expression in −/− and WT T_regs are displayed below the corresponding band in the bar graph. (D) Representative CD25 versus FoxP3 staining after gating on CD4⁺CD8⁻ thymic cells from SHIP^ΔIP/ΔIP or SHIP^−/− and WT littermate controls. (E) Percentage frequency of CD25⁺FoxP3⁺ and CD25⁻FoxP3⁺ T_regs after gating on CD4⁺CD8⁻ T cells and total absolute number of CD4⁺CD25⁺FoxP3⁺ and CD4⁺CD25⁻FoxP3⁺ T_regs in the thymus of the indicated genotype. For SHIP^ΔIP/ΔIP mice: n = 5 and littermate control: n = 5. For SHIP^−/− mice: n = 4 and littermate control: n = 4. *P < .05. **P < .01. ***P < .001.