Impaired AP-1 promoter DNA-binding in naturally developed Tregs. (A) Purified CD4⁺ CD25⁺ regulatory T cells, with CD4⁺ CD25⁻ naive T cells as controls, were stimulated with anti-CD3 or anti-CD3 plus anti-CD28 for 36 hours. Nuclear extracts were isolated from these stimulated cells and used for electrophoretic mobility shift assay assay using biotin-labeled DNA fragments corresponding to the promoter region of AP-1 (top panel), NF-AT (middle panel), or NF-κB (bottom panel). (B) The bands' density of AP-1 DNA-binding was measured using National Institutes of Health 1.63 software. Error bars represent data from 5 independent experiments. Student t test was used for the statistical analysis. (C) The protein expression of FoxP3, c-Jun, c-Fos, and JunB from the nuclear extracts used in panel A was analyzed in parallel by Western blotting.