FIGURE 1.

A, FACS analysis of purified CD4⁺CD25⁺ cells for FoxP3, active GSK-3β, and phosphorylated β-catenin. Single cell suspensions of isolated CD4⁺CD25⁺ cells from B6 and BALB/c spleen and lymph nodes demonstrate co-staining of FoxP3⁺, GSK-3β (Tyr(P)-216), and phosphorylated β-catenin (Ser(P)-33/Ser(P)-37/Thr(P)-41). This finding demonstrates that CD4⁺CD25^+hiFoxP3⁺ cells exhibit high GSK-3β activity. Moreover, the signal overlaps with CD4⁺CD25^+hi cells stained for β-catenin (Ser(P)-33/Ser(P)-37/Thr(P)-41), suggesting that CD4⁺CD25^+hiFoxP3⁺ cells possess elevated GSK-3β activity, reflected in increased β-catenin phosphorylation. Panels A–C, B6 spleen; panels D–F, B6 lymph nodes; panels G–I, BALB/c spleen. Data are representative of at least three independent experiments, gating on the purified lymphocyte population. B, histogram analyses of purified CD4⁺CD25⁻ and CD4⁺CD25⁺ cells for phosphorylated β-catenin (Ser(P)-33/Ser(P)-37/Thr(P)-41). This finding demonstrates that CD4⁺CD25^+hi cells exhibit higher GSK-3β activity than CD4⁺CD25⁻FoxP3⁻ cells. Panel A, B6 spleen; panel B, B6 lymph nodes; panel C, BALB/c spleen. Shaded area, CD4⁺CD25^+hi cells plus Alexa Fluor 488-labeled secondary antibody alone; dashed line (---), CD4⁺CD25⁻FoxP3⁻ cells plus Alexa Fluor 488-labeled secondary antibody alone; solid line (—), CD4⁺CD25^+hi cells plus anti-phosphorylated β-catenin (Ser(P)-33/Ser(P)-37/Thr(P)-41) antibody; dotted line (···), CD4⁺CD25⁻FoxP3⁻ cells plus anti-phosphorylated β-catenin (Ser(P)-33/Ser(P)-37/Thr(P)-41) antibody. Data are representative of at least three independent experiments, gating on the purified, lymphocyte population.