FoxP3 alters c-Jun subnuclear localization. (A,B) c-Jun-CFP expression plasmids were transfected without (A) or with (B) FoxP3 expression plasmids into HEK 293 cells. Transfected cells were immunostained with anti-FoxP3 antibody followed by a second antibody that has been labeled by Texas-Red. The nuclear DNA was stained with DAPI (blue). Cells were then visualized under the fluorescence microscopy. (C,D) CD4⁺ CD25⁺ cells and CD4⁺ CD25⁻ cells were purified and stimulated with anti-CD3 and CD28 for 24 hours. Cells were cyto-spun onto slides, fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and immunostained with anti-FoxP3 (rat) and anti–c-Jun (rabbit), followed by anti–rat-Alexa 594 (red) and anti–rabbit-Alexa 488 (green). The nuclear DNA was stained with DAPI (blue). Cells were then visualized under the fluorescence microscopy and representative images were selected.