Inhibition of NFAT activity reduces BLyS protein expression in NHL-B cells. (A) EMSA analysis of NFAT protein binding to BLYS promoter. Nuclear extracts from NHL-B (LBCL-MS) cells were transfected with dominant-negative (DN) NFAT or control plasmid and analyzed with oligonucleotide probes from the 2 BLYS-NFAT binding sites. NFAT binding levels were analyzed by BioMax 1D software, presented as before, as relative fold decrease compared to control samples. Lamin B was used as nuclear protein loading control. (B) Immunoblot analysis of BLyS expression in NHL-B (LBCL-MS) cells transfected with DN NFAT or control plasmid. Actin is used as loading control. BLyS protein levels were analyzed by BioMax 1D software, normalized with actin levels as before, and presented as before. (C) EMSA analysis of NFAT binding to the BLYS promoter in a representative NHL-B cell line (MCL-SP53). Nuclear extracts from NHL-B (MCL-SP53) cells treated with cyclosporine A (CsA) were analyzed with oligonucleotide probes from the 2 BLYS-NFAT sites to detect NFAT binding activity. NFAT binding levels were analyzed as previously described in panel A. Similar results observed in LBCL-MS cells too (data not shown). (D) Immunoblot analysis of BLyS protein in NHL-B (MCL-SP53) cells treated various doses of CsA. Actin was used as loading control. BLyS protein expression levels were analyzed as previously described in panel B. Similar results observed in LBCL-MS cells too.