NFATc1 and NF-κB c-rel directly interact and synergistically regulate CD154 gene transcription in LBCL cells. (A) LBCL-MS cells were fixed with methanol and stained for NFATc1 (green), c-rel (red), and nuclear marker Topro (blue) and analyzed by confocal immunofluorescence analysis. Colocalization of NFATc1 and c-rel appears yellow. (B) Nuclear extracts from LBCL-MS and LBCL-EJ cells were immunoprecipitated with a polyclonal c-rel antibody or IgG antiserum (negative control). Immunoprecipitated complexes were resolved on SDS–polyacrylamide gel electrophoresis (PAGE), and subjected to Western blotting with anti-NFATc1 or anti–c-rel antibody. NE indicates 25 μg of nuclear extract from MS cells. (C) Because the NFATc1 antibody was poor for immunoprecipitation, LBCL-MS and LBCL-EJ cells were transfected with the Flag-tag-NFATc1 expression vector, and their nuclear extracts were used for coimmunoprecipitation with anti-Flag antibodies and immunobloting with c-rel or NFATc1. NE indicates 10 μg of nuclear extracts from MS cells. (D) LBCL-MS cells were cotransfected with CD154p-luc reporter and the expression vector for c-rel (pc-rel, 10 μg), NFATc1 (pNFATc1, 10 μg), or both. The pGL3 empty reporter plasmid was used as a negative control. After 24 hours, luciferase activity was determined and corrected for transfection efficiency using β-gal activity. The data are representative of 3 independent experiments. The error bars indicate the standard deviation of triplicate samples. (E) Whole-cell lysates from panel D were also analyzed for NFATc1, c-rel, and actin protein expression. Actin was used as an internal loading control. (F) Transfected cells from panel D were also used to obtain total RNA for analysis of CD154 mRNA by RT-PCR. GAPDH transcripts were measured to indicate equivalent amounts of RNA used for each reaction.