2.4. Electrophoretic Mobility Shift Assay (EMSA)

To assess NF-κB binding to promoter sequences, nuclear extracts from RAW 264.7 macrophages were prepared, according to the method of Staal et al. (1990) and subjected to electrophoretic mobility shift assays (EMSAs) using the biotin end-labeled double-started NF-κB probe (5′-biotin-AGTTGAGGGGACTTTCCCAGGC-3′). Nuclear protein extract (4 mg) was incubated on ice for 1 h with 0.25 pmole ³²P-end-labelled oligonucleotide in binding buffer containing 20 mM HEPES (pH 7.5), 4% Ficoll, 0.5 mg/mL poly-DIDC, 0.1 mM MgCl₂, and 0.1 mM DTT. The nuclear extract complexed with NF-κB probe was separated from free oligonucleotides by 4% non-denaturing polyacrylamide gel electrophoresis in TBE buffer (89 mM Tris-HCl, pH 8.0, 89 mM boric acid, and 2 mM EDTA). The biotin–DNA complex was detected using the enhanced LightShift Chemiluminescent EMSA Kit (Panomics, Fremont, CA, USA) and visualized using a CAS-400SM Davinch-Chemi chemiluminescence imaging system (Seoul, Korea).