Fig. 5

NFAT binds to the RORC locus in stimulated CD4⁺ T cells. a NFATc1 binds to the RORC locus in stimulated CD4⁺ T cells. ChIP was performed on cord blood CD4⁺ T lymphocytes, followed by RT-qPCR of the indicated genomic regions. b NFATc2 binds to the RORC locus in Th17 cells. ChIP was performed as in a. Shown are one representative experiment of 3, average of three technical replicates. c Left: human/murine alignment of the RORC2 promoter and upstream conserved region (CNS-2.5 kb and -1.8 kb), with the position of the predicted NFAT binding sites tested in the DNA affinity capture assay. Right: DNA affinity capture assay on lysates from Jurkat E6.1 cells stimulated with PMA/ionomycin for 30 min. The western blot of DNA-bound proteins was probed with anti-NFATc2 antibody. IL-2 prom: characterized NFAT site in the IL-2 promoter. neg: sequence in the RORC locus with no predicted NFAT binding sites. d DNA affinity competition assay. Jurkat E6.1 lysates were incubated with biotinylated NFAT binding oligos, together with non-biotinylated oligos containing a wild-type (W) or mutated (M) NFAT binding site. e DNA affinity capture assay on cord blood CD4⁺ T cells stimulated in the indicated conditions (“−”: unstimulated, TT: TCR + TGFβ; 17: Th17 polarizing condition). f Activity of the RORC2 promoter in cell lines. HEK 293 T and Jurkat E6.1 cells were transfected with the indicated reporter constructs. Normalized luciferase activity is represented as fold change over the activity of pGL3 basic. Average and SD of 4 experiments. g NFATc2 activates transcription from the RORC2 promoter. HEK293T cells were transfected with the RORC2 promoter reporter and the shown NFAT constructs. Luciferase activity is represented as fold change relative to activity of the RORC2 promoter alone (first bar). Average and SD from 15 transfections. Two-tailed t-test was performed as shown. Source data are provided as a Source Data file