Positively charged nylon membrane

Nuclear extracts were prepared using the NE-PER nuclear and cytoplasmic extraction reagents as described above. Synthetic complementary NF-κB-binding (5′-AGTTGAGGGG ACTTTCCCAGGC-3′) and Nrf2-binding (5′-GCTCTTCCGGTGCTCTTCCGGT-3′) oligonucleotides (Affimetrix Inc., CA, USA) were 5′-biotinylated using a biotin 5′-end DNA labeling EMSA kit (Affimetrix Inc.) according to the manufacturer’s protocol. The binding reactions contained 10 μg of nuclear extract proteins, binding buffer, 1 μg of poly d(I-C), and 10 ng of biotin-labeled DNA. The reactions were incubated for 5 min at room temperature in a final volume of 10 μl. The protein-DNA complexes were separated from the DNA probes by electrophoresis on a native 6% polyacrylamide gel that had been pre-electrophoresed for 1 h in 0.5× Tris borate/EDTA buffer (50 mM Tris base, 18 mM boric acid, 500 mM EDTA, pH 8.3). Complexes were then transferred to a positively charged nylon membrane (Pall Corporation, Pensacola, FL, USA) in 0.5× Tris borate/EDTA buffer at 300 mA for 30 min, after which the DNA was hybridized to the nylon membrane in a drying oven at 80°C for 1 h. Horseradish peroxidase-conjugated streptavidin was used according to the manufacturer’s instructions to detect the transferred DNA.