PMA/ionomycin-inducible and CsA-sensitive NFAT binding in human LTα promoter.
(A) Sequence of human LTα promoter. Predicted NFAT and NFκB sites are in bold, sequences of the corresponding oligonucleotide probes are underlined. (B) Binding to NFAT probes. PBMCs were treated with 100 ng PMA and 2 μM ionomycin for 10 minutes (P/I) in the presence (+) or absence (−) of 400 nM CsA. CsA was added 30 minutes prior to activation. Nuclear extracts from 1 to 3 × 107/mL PBMCs were analyzed in EMSA with different predicted NFAT-binding probes from human LTα promoter. Specificity of PMA/ionomycin-inducible and CsA-sensitive NFAT binding to NFAT4 site was confirmed by abolishing the binding with anti-NFAT, but not anti-NFκB1 sera. (C) NFκB binding. Brackets indicate the location of NFAT (B) or NFκB (C) complexes. *NS indicates nonspecific bands.
