FoxP3 mRNA and protein expression. (A) Levels of FoxP3 mRNA were assessed by real-time PCR analysis. Samples include freshly isolated cells, cell lines after 4 weeks of culture, and 4 week–cultured cell lines 24 hours after restimulation with anti-CD3/CD28 beads, as indicated. Data are plotted as fold comparison to mRNA levels present in freshly isolated CD8⁺ T cells. CD4⁺CD25^--derived cells and lines are indicated by blue bars and CD4⁺CD25⁺ cells and lines by burgundy bars. (B) Western blot analysis of FoxP3 protein expression in cultured cells. Cell lines were cultured for 4 weeks and restimulated with anti-CD3/CD28 beads for 15 minutes, 2 hours, or overnight. The level of histone H3 was used as a loading control for nuclear proteins. Results representative of 4 independent experiments.