(A) Flow cytometry plots and cumulative data (mean ± SEM) from individual mice (n = 4–6/group) depicting the percentages of thymic Thy1.2⁺CD3⁺CD4⁺CD8^–-SP FOXP3-YFP⁺ CD4⁺ Tregs that are PP2A deficient (KO) or PP2A sufficient (WT). A representative of 3 experiments is shown. (B and C) Thymic CD4⁺ Tregs (defined as Th1.2⁺CD3⁺CD4⁺CD8^–-SP Foxp3-YFP⁺ cells) were sorted from individual KO and WT mice (n = 5/group). (B) Total RNA from thymic CD4⁺ Tregs from individual KO and WT mice (n = 4–5/group) from 2 experiments was assessed for mRNA transcripts of Foxp3. Individual and cumulative data (mean ± SEM) are shown. (C) Protein lysates were prepared following the pooling of cells in each group. The expression of Foxp3 and β-actin was assessed by Western blot analysis (right), and the Foxp3/β-actin ratio (mean ± SEM) was calculated from 2 independent experiments (left). (D) Expression of CD25 in thymic CD4⁺ Tregs from the KO and WT mice. Representative histograms (left) and cumulative data (right) from a representative of 3 experiments are shown (mean ± SEM). Dashed-line histograms represent negative control. (E) Thymic precursors of CD4⁺ Tregs defined as Thy1.2⁺CD4⁺CD8^–CD25^hiFOXP3-YFP^– cells were sorted from KO and WT mice (n = 5/group) and challenged with or without IL-2 (20 mM) in plates precoated with α-CD3 for 18 to 24 hours. Thereafter, the cells were pooled in each group and protein lysates were prepared. The expression of Foxp3 and β-actin was assessed by Western blot analysis (top), and the Foxp3/β-actin ratio (mean ± SEM) was calculated from 2 independent experiments (bottom). *P < 0.05; **P < 0.005.