(A) Relative mRNA expression amounts of Tbx21, Gata3, c-Maf and Rorc in sorted CD4⁺YFP⁺ Treg cells from Itch^fl/flFoxp3^Cre mice and Itch^+/+Foxp3^Cre littermates. Results of three experiments are shown as the mean ± SD values. (B) FACS-sorted CD4⁺ YFP⁺ Treg cells were fixed and stained with either anti-GATA3 antibody or an isotype-matched control Ig. For phospho-STAT6 detection (p-STAT6), FACS-sorted CD4⁺YFP⁺ Treg cells were stimulated with anti-CD3/CD28 antibodies for 2 hours, followed by staining with antiphospho-STAT6 antibody. (C) Immunoblot analysis of wild-type and Itch-deficient Treg cells. CD4⁺YFP⁺ Treg cells sorted from Itch^fl/flFoxp3^Cre and Itch^+/+Foxp3^Cre mice were stimulated with anti-CD3/CD28 antibodies for 24 hours. Cell lysates were separated by SDS-PAGE and immunoblotted with the indicated antibodies. The blot for Itch is the same as that in Figure 1A. (D) Flow cytometric analysis of YFP and mAmetrine expression in CD4⁺ T cells in the bone marrow chimeric mice (BMC). shRNA-expressing bone marrow chimeric mice were generated by reconstituting retrovirally transduced bone marrow cells from Itch^fl/flFoxp3^Cre mice into lethally irradiated recipient mice. YFP and mAmetrine double-positive CD4⁺ T cells were sorted and activated with anti-CD3/CD28 antibodies for 2 days in vitro. Cytokine concentration in the culture supernatants was measured by an ELISA assay. Data are compiled from two independent experiments with four mice per group. Error bars indicate the mean (± SD). *P < 0.05; **P < 0.01; ***P < 0.001.