NFAT cooperates with NF-κB to regulate CD154 transcription in LBCL cells. (A) LBCL-MS cells were cotransfected with CD154p-luc (10 μg) and the expression vector DN-NFAT (10 μg), DN-IκBαM (10 μg), or the empty control vector pCDNA3 along with 1 μg of pCMV–β-gal. After 24 hours, luciferase activity was determined and corrected for transfection efficiency using β-gal activity. Transfected cells from panel A were also used to obtain purified total RNA for analysis of CD154 mRNA by RT-PCR, cytoplasmic extracts for analysis of CD154 protein expression (B), and nuclear extracts for analysis for CD154-κB and NFAT2 DNA binding by EMSA (C). GAPDH, actin and Oct-1 were used as internal loading controls for panels B and C, respectively. (D) Nuclear extracts from LBCL-MS cells were incubated with the p³²-labeled NFAT2, CD154-κB, or Oct-1 probe along with monoclonal (M) or polyclonal (R) antibody against c-rel, NFATc1, or Oct-1, and analyzed by EMSA. (E) Nuclear extracts from LBCL-MS cells were incubated with antibody against c-rel, NFATc1, or Oct-1 alone or combined and analyzed by EMSA. (F) ChIP analysis was performed on LBCL-MS cells after transfection with a Flag-NFATc1 expression vector. The indicated primary antibodies were used to precipitate chromatin after cross-linking. PCR to detect the CD154 promoter regions (CD154-κB or NFAT2) was performed on the precipitated DNA. The full-length CD154-luc reporter plasmid was used as a positive control. IgG was used as a nonspecific antibody control.