NFAT proteins bind to the human c-FLIP promoter in activated T cells. (A) HuT78 cells were stimulated for the indicated time with 20 ng/mL PMA and 1 μM ionomycin with or without 1 μM CsA or 10 μg/mL cycloheximide (CHX). c-FLIP expression was analyzed by Western blotting. Tubulin served as a loading control. (B) HuT78 cells were treated for the indicated time with 20 ng/mL PMA and 1 μM ionomycin with or without 1 μM CsA. EMSAs were performed with radioactive oligonucleotides comprising either the putative NFAT binding site 1 or site 2 of the human c-FLIP promoter. ▶ indicates the NFAT-specific DNA complex. (C) For competition assays, nuclear extracts of PMA/ionomycin-stimulated cells were incubated with the radioactively labeled NFAT consensus oligonucleotide and the indicated relative concentrations of unlabeled site 1 or site 2 oligonucleotides. Site 1 and site 2 oligonucleotides with a mutation in the putative NFAT recognition sequences were used as negative controls. As a negative control, oligonucleotide incubated without nuclear extract (no lysate) was loaded. A nuclear extract from stimulated cells in the absence of competing oligonucleotide (w/o comp) served as a positive control. (D) Supershift analyses were performed by incubation of nuclear extracts of PMA/ionomycin-stimulated cells with NFATc1, NFATc2, NFATc3, c-Jun, or a control mouse IgG (mIgG) antibody. The NFAT-specific and supershifted DNA/protein complexes are indicated by ▶ and ▷, respectively. (E) Human peripheral T cells were stimulated with 5 μg/mL PHA-L for the indicated time points. Nuclear extracts were prepared and EMSA analyses were performed using either the putative NFAT site 1 or site 2 oligonucleotides. (F) Freshly prepared human peripheral T cells were incubated with PHA-L or PHA-L plus CsA for 16 hours. EMSA analyses were performed by incubation of nuclear extracts with radioactive NFAT site 1 or site 2 oligonucleotides.