NF-κB p65 up-regulates expression from the MIR155HG promoter through an NF-κB binding site located upstream of the transcription start site. (A) At least three NF-κB binding sites are predicted in sequences within 1494 nt upstream of the MIR155HG transcription start site (+1): the -1150 site was previously reported  and the sites at -441 and -178 were predicted using the online prediction programs Promo and TRED. (B) Reporter assays were performed in COS-1 cells transfected with the pGL3-based wild-type MIR155HG promoter luciferase reporter plasmid (WT-MIR155HG) and either pcDNA alone (-), pcDNA-p65, pcDNA-REL, or pcDNA-p50. Relative luciferase values for each transfection were normalized for RSV-renilla expression and then normalized to the value for pcDNA alone (1.0), +/- SE, and the * indicates p < 0.2E-8. (C) The pcDNA versions of p65, REL, and p50 (used in B) were expressed individually in COS-1 cells and whole cell extracts were prepared. Western blotting for p65, REL, p50, and β-tubulin (normalizing control) confirmed expression of all three proteins. (D) Site-directed mutagenesis was performed to create mutants of each predicted NF-κB binding site, creating -1150mut-MIR155HG, -441mut-MIR155HG, and -178mut-MIR155HG reporter plasmids. Transfections and luciferase assays were performed as in (B). The * indicates p < 0.002.