Cells were washed in PBS, scraped, and suspended in cytoplasmic extract buffer [10 mM Hepes (pH 7.6), 60 mM KCl, 1 mM EDTA, 0.25% NP-40, and protease inhibitor cocktail]. Nuclei were subsequently extracted with 20 mM tris-HCl (pH 8), 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, and 25% glycerol. Nuclear extracts were incubated with poly(dI-dC) for 20 min at room temperature with 32P-labeled probe [NF-κB: 5′-CAGGGCTGGGGATTCCCCATCT-3′ (58), SP1/SP3: 5′-ATTCGATCGGGGCGGGGCGAGC-3′ (59), IRF1: 5′-GGAAGCGAAAATGAAATTGACT-3′ (60)]. In some cases, nuclear extracts were preincubated with 100-fold excess of cold wild-type or mutant competitor probes for 5 min at room temperature (mutant NF-κB: 5′-CAGGGCTGCGGCTTCCCGATCT-3′). Samples were resolved on a 5% polyacrylamide gel and exposed to film. EMSA supershift was performed by incubating nuclear extracts with 1 μg of the following antibodies at room temperature for 15 min: IgG (2027), SP1 (14027), SP3 (644), or IRF1 (497) from Santa Cruz Biotechnology.

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