NFATc1 binds to the IL-17 promoter in TLo, but not THi, cells. Human CD4⁺ T cells were cultured under THi or TLo conditions for 6 days. Day 6 cells were either restimulated for 2 hours with PMA/ionomycin (+) or left untreated (-). (A) Cytoplasmic and nuclear lysates were prepared, and expression of NFATc1 was determined by Western blotting. Blots are representative of 3 independent experiments. (B) NFATc1 binding to the proximal IL-17 promoter was assessed by ChIP (n = 2). Error bars respresent SEM. (C) CD4⁺ T cells were cultured for 6 days under either THi or TLo conditions or TLo conditions +500nM ionomycin (TLo +) added daily for the first 4 days of culture. Expression of IL-17 was determined by intracellular flow cytometry (n = 3). Error bars represent SEM.