PMC:3317373 / 5456-7257 JSONTXT

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Protein concentration was determined using a bicinchoninic acid assay kit with bovine serum albumin as the standard (Pierce Biochemicals, Rockford, IL, USA). EMSA was performed using gel shift assay system (Promega, Madison, WI, USA). Consensus oligonucleotide probe (5′-AGTTGAGGGGACTTTCCCAGGG-3′) was end-labeled by T4-polynucleotide kinase using [γ-32P]-ATP (Free Biotech., Beijing, China). Nuclear protein (20 μg) was preincubated in 20 μL binding buffer containing 10 mM Tris-HCl (PH 7.5), 1 mM MgCl2, 0.5 mM NaCl, 4% glycerol, 0.5 mM EDTA, 0.5 mM DTT, and 2 μg poly dI-dC for 20 minutes on ice. After addition of the 1 μL 32P-labled oligonucleotide probe, the incubation was continued for 20 minutes on ice. The DNA-protein complexes were separated by electrophoresis on 4% nondenaturing polyacrylamide gel in 0.5 × TBE buffer (tris-borate-EDTA) at 390 V for 1 hour at 4°C. After electrophoresis, the gel was dried and exposed to X-ray film (Fuji Hyperfilm, Tokyo, Japan). 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