FIGURE 2.

Physical association of NFAT1 with the IL-9 promoter. A, ChIP assay was performed with PMA/ionomycin-stimulated Th1 and Th9 cells using control IgG and NFAT1 antibody. The amounts of precipitated DNA were measured by quantitative PCR with primers specific for the IL-9 and IFN-γ promoter regions and are represented relative to their amount in total chromatin (input) as a fraction of input. B, left, antibodies against NFAT1 and actin (control) were used to perform Western blot for detecting NFAT1 expression in untransfected and NFAT1-transfected HEK cells. Right, biotin-conjugated probe corresponding to NFAT binding site (at −48/−38 in Fig. 1A; NFAT-e) were incubated with NFAT1-overexpressing HEK-293 cell lysate in the presence of the indicated non-biotinylated competitor probes. Cons and mt-Cons, conserved NFAT binding and NFAT nonbinding mutant probes, respectively. The protein DNA complexes were precipitated with streptavidin (Strep) and analyzed by immunoblotting with α-NFAT1 antibody. The first lane indicates input, which is 2.5% of the total cell extract (CE) used for pull-down. The data are representative of three independent experiments. C and D, HEK-293 (C) or EL-4 cells (D), respectively, were transfected with empty control (mock) or luciferase reporter constructs containing IL-9 promoter (IL-9 Pro; encompassing the −366/+48 region as shown in Fig. 1A) or enhancer containing IL-10 promoter (IL-10 CNS-9 (30)) in the presence of NFAT1 expression vector. Cells were stimulated with PMA/ionomycin for 8 h or with cyclosporin A (CsA) 20 min prior to PMA/ionomycin stimulation, and the luciferase assay was conducted. The luciferase activity was calculated relative to the activity of Renilla luciferase and represented in relative luciferase units (RLU) as a -fold difference relative to the control (mock/empty plasmid) value. The data shown are expressed as mean ± S.E. (error bars), n = 3; **, p < 0.01; ***, p < 0.001.