The NF-κB p50/p65 heterodimer binds to the -178 site from the MIR155HG promoter to activate transcription. (A) FLAG-tagged versions of p65 and p50 were expressed individually or together in A293 cells and whole cell extracts were prepared. Western blotting for FLAG, p65, p50, and β-tubulin (normalizing control) confirmed expression of p65 and p50. (B) An EMSA was performed using the extracts from (A) with a probe containing -178 site from the MIR155HG promoter (see Figure 2). Lanes are as follows: probe alone (lane 1), empty vector (lane 2), p65 alone (lane 3), p50 alone (lane 4), p50/p65 (lane 5), p50/p65 with 50X cold probe competition (lane 6), p50/p65 with pre-immune serum (lane 7), p50/p65 with SC-372x p65 antibody (lane 8), and p50/p65 with NR-1226 p65 antibody (lane 9). The supershifted p50/p65 complex is indicated by ss and an arrow. (C) An EMSA was performed using extracts from (A) with a consensus NF-κB site from the human MHC1 enhancer (as a κB-site control [32, 33, 38, 50]) and complexes were detected by autoradiography. The appearance of single bands in the p65 and p50 alone lanes confirms that both proteins can bind DNA. Upon co-expression of p65 and p50, a band between the p65 and p50 bands is present, representing the p50/p65 heterodimer. (D) Reporter assays were performed in mouse 3T3 and 3T3nfkb1-/-nfkb2-/-relb-/-crel-/- (4KO) cells transfected with the WT-MIR155HG reported plasmid and pcDNA expression plasmids for either vector control (-), FLAG-p65, or FLAG-p50. Values for each transfection were normalized to RSV-renilla and then normalized to pcDNA alone (1.0), +/- SE. The * indicates p < 0.03, and n.s. indicates that the difference is not statistically significant.