An NFAT-containing protein complex binds to a putative NFAT site in the A1 proximal promoter. (A) Sequences of the wild-type (wt) and mutated (mut) oligonucleotide probes used in the EMSA with the putative binding site for NFAT indicated in bold and the 3 base pair mutation in italics. (B) EMSA on nuclear extracts from C57 cells activated by IgE receptor crosslinking or treatment with ionomycin (1 μM). Activation of C57 cells results in an increased binding of a nuclear protein complex to the oligonucleotide probe shown in panel A. This complex formation is abrogated by pretreatment with unlabeled competitive wt but not mutated oligonucleotide probe. Mutated oligonucleotide probe does not form binding complex at all. (C) The augmented complex binding from activated C57 is inhibited by pretreatment of the cells with cyclosporin A. (D) NFAT-specific antibodies (anti-NFAT = NFATc1; anti-NFAT1 = NFATc2) cause a band shift in the EMSA, indicating the presence of NFAT within the DNA-protein complex. Representative results from 2 independent experiments are shown.