Constitutive activation of NFAT is required for CD154 expression in LBCL cells. (A) NFAT sites NFAT1 (-74 to -56), NFAT2 (-270 to -252), and NFAT3 (-768 to -750) on the CD154 promoter were synthesized and used as probes for EMSA with nuclear extracts from tonsillar B cells (TB), LBCL-MZ cells, and LBCL-MS cells. (B) Nuclear extracts from LBCL-MS cells were incubated with NFAT2 cold probe, AP-1 cold probe, NF-κB cold probe, or antibody against NFATc1, NFATc2, NFATc3, NFAT4, NFAT5, or Oct-1, and analyzed by EMSA with the NFAT2 p³²-labeled probe. FP indicates free probe alone. (C) Nuclear extracts (50 μg) from normal B cells (NB), normal T cells (NT), LBCL cell lines, and primary LBCL cells from patients were analyzed by Western blot for NFATc1 expression. As indicated (- or +), cells were stimulated with PMA (20 ng/mL) and ION (1 μM). Lamin B indicates equal loading of nuclear extracts. (D) Control LBCL-MS cells and cells treated with FK-506 (5 μg/mL) or CsA (5 μg/mL) were fixed with methanol, stained for NFATc1 (red) and nuclear marker TOPRO-3 (blue), and analyzed by confocal immunofluorescence microscopic analysis. (E) Nuclear extracts from cells from panel D were analyzed for NFAT2 DNA binding by EMSA analysis. Ethanol was used as a control vehicle. Oct-1 DNA binding was used as an internal loading control.