The human c-FLIP promoter is induced upon NFAT activation. (A) Schematic representation of the luciferase reporter construct comprising the human c-FLIP promoter I (in gray). The reporter construct contains both putative NFAT binding sites indicated as □. (B) Luciferase assays were performed in 293T cells transiently transfected with the c-FLIP reporter construct (pGL3-FLIP), an empty vector control (pGL3), or, as a positive control, a reporter construct comprising 3 tandem repeats of the NFAT binding site of the IL-2 promoter (NFAT). Expression plasmids encoding either NFATc1 or NFATc2 and constitutively active calcineurin were cotransfected. At 24 hours after transfection, luciferase activity was quantified. β-galactosidase activity was used for normalization. Results are displayed as the mean plus or minus SD of 3 independent experiments. (C) ChIP analysis of the c-FLIP promoter in CEM cells stimulated for the indicated time points with 20 ng/mL PMA and 1 μM ionomycin. ChIP procedure was done as described in “Chromatin immunoprecipitation.” NFAT-chromatin complexes were immunoprecipitated with NFATc2 and NFATc1 antibody, respectively. Rabbit IgG (rbIgG) and mouse IgG (msIgG) were used as negative controls. PCR was performed with primers specific for the NFAT binding site 1 of the c-FLIP promoter and analyzed by agarose gel electrophoresis. Genomic DNA (input) was used as a positive control in the PCR analysis.