Evaluation of protein-DNA complex formation on the putative NF-κB motifs by EMSA. The sense (as listed in Figure 2 legend) and antisense synthetic oligonucleotides representing either wild-type or single mutant NF-κB sequences were annealed and 32P-labeled to form the double-stranded DNA probes. The 32P-labeled double-stranded 20-bp oligonucleotide probes were incubated with 7.5 μg PMA-stimulated Jurkat (A) or HUT78 (B,C) for 30 minutes at room temperature (RT). For antibody supershift analysis, nuclear extracts were incubated for 10 minutes at RT with 1 μL antibody (Santa Cruz Biotechnology) specific for NF-κB p65 (sc-372x, C-term; and sc-109x, N-term), NF-κB p50 (sc-7178x), or IRF-2 (sc-498x) prior to addition of the 32P-labeled wild-type, mutant NF-κB1, or NF-κB2 probes as indicated at the bottom of each panel. For oligonucleotide competition experiments, 100-fold molar excess of indicated unlabeled probes was added to each binding reaction. The DNA-protein complexes were resolved on 4% native PAGE and exposed to KODAK X-Omat LS films (Rochester, NY). A vertical line has been inserted into panel B to indicate where gel lanes were cut. Note that all images in panel B came from the same experiment. *indicates absence of NF-κB protein-DNA complex in lane 11 when a mutant probe is used.
