2.8. Nuclear Protein Extraction and NF-κB Electrophoretic Mobility Shift Assay (EMSA)

The ICB tissues and RAW 264.7 cells were trypsinized, resuspended, and homogenized in buffer A [10 mM HEPES (pH 7.9), 1.5 mM KCl, 10 mM MgCl₂, 1.0 mM dithiothreitol (DTT), and 1.0 mM phenylmethylsulfonyl fluoride (PMSF)]. The tissue was then homogenized (Dounce; Bellco Glass Co., Vineland, NJ), followed by centrifugation at 5000 ×g at 4°C for 10 min. After suspension in 200 mL of buffer B [20 mM HEPES (pH 7.9), 25% glycerol, 1.5 mM MgCl₂, 420 mM NaCl, 0.5 mM DTT, 0.2 mM EDTA, 0.5 mM PMSF, and 4 μM leupeptin], the sample was incubated on ice for 30 min and centrifuged at 12000 ×g at 4°C for 30 min. The supernatant containing the nuclear proteins was collected and stored at −70°C until use.

A bicinchoninic acid assay kit, with bovine serum albumin as the standard (Pierce Biotechnology, Rockford, IL), was used to determine the protein concentration. EMSA was performed with an NF-κB DNA-binding protein-detection system (Pierce Biotechnology) according to the manufacturer's instructions. We incubated a 10 μg nuclear protein aliquot with a biotin-labeled NF-κB consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGC-3′) for 30 min in binding buffer and then determined the specificity of the DNA/protein binding by adding 100-fold molar excess of unlabeled NF-κB oligonucleotide for competitive binding 10 min before adding the biotin-labeled probe.