Electrophoretic mobility shift assay (EMSA) and supershift assay

EMSA was performed based on the methods published previously with slight modifications⁶². Nuclear extract (20 μg of protein amount) was incubated with 20 µl 0.01 μM NF-κB-specific oligonucleotide as described above in 5 mM Tris–HCl (pH 8.0) containing 50 mM NaCl, 0.05% Nonidet P-40, 1 mM MgCl₂, 0.5 mM DTT, 5% (v/v) glycerol, 0.25 mM EDTA, 40 ng/ml BSA, and 0.2 mg/ml poly (deoxyinosinic-deoxycitidylic) acid sodium salt for 30 min at room temperature. Subsequently, the reaction mixture (15 µl) was loaded on 6% native polyacrylamide gels in 1 × Tris–borate-EDTA buffer (Nacalai Tesque, Kyoto, Japan). Electrophoresis was performed at 150 V for 60 min at 4 °C. Bands of 6-FAM-labeled DNA on the gels were detected with FLA-5100 (FUJIFILM Corporation, Tokyo, Japan) using an excitation wavelength of 473 nm and emission wavelength of 510 nm. Supershift assay was performed using antibodies against NF-κB subunits: p65, RelB, c-Rel, p50, and p52. Nuclear extract (20 μg of protein amount) was incubated with 5 μg of each antibody for 30 min at room temperature in a total volume of 10 µl. Vehicle control was incubated with PBS containing 0.1% (w/v) NaN₃. EMSA was performed as described above except that the electrophoresis was performed for 70 min.