CD154 promoter deletions and site-directed mutagenesis. (A) LBCL-MS cells were transfected with equivalent concentrations of the indicated CD154 promoter constructs (10 μg each) or cotransfected with the promoter constructs and the expression vector for c-rel and NFATc1 (10 μg each). After 24 hours, luciferase activity was determined and corrected for transfection efficiency using β-gal activity. Numerals at left of constructs indicate base pairs. (B) LBCL-MS cells were cotransfected with the 1200-bp wild-type CD154 reporter construct or with the 1200-bp CD154 construct with the mutated κB site, mutated NFAT2 site, or both (10 μg) and 10 μg each of c-rel and NFATc1 expression plasmids. After 24 hours, luciferase activity was determined and corrected for transfection efficiency using β-gal activity. The data in panels A and B are representative of 3 independent experiments. (C) Specificity binding of proteins to CD154-κB and NFAT2 sites. Nuclear extracts from LBCL-MS cells were incubated with wild-type (wt) or mutant (mut) κB, NFAT2 p³²-labeled probes along with the indicated cold probes (cp), and analyzed by EMSA.