Transcriptional regulation of the CD40 gene by NF-κB.

(A) Schematic map of the CD40 promoter and mutant constructs. Consensus NF-κB binding sites are designated NF-κB1, NF-κB2, and NF-κB3. Mutant NF-κB3 is shown in gray. The transcriptional start site is indicated corresponding to the previously published mRNA sequence.46 (B) Reporter gene activation. COS-7 cells were transfected with 200 ng of the different reporter constructs and 200 ng of p50/p65 expression constructs or pCDNA3 to give a total of 400 ng; 100 ng pRL-TK was cotransfected in each reaction. Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). (C) TNF-α stimulates CD40 expression via NF-κB. Control and IκBΔN–transfected Namalwa cells were treated with TNF-α (25 ng/mL). NF-κB DNA binding activity in whole-cell extracts was determined by EMSA (upper panel). Flow cytometric analysis of surface CD40 expression. Cells were either untreated or treated for 20 hours with TNF-α (lower panels).