Poly d 1 c

The oligonucleotide (Table 1) was labeled with fluorophore Cy3 and annealed with complementary oligonucleotide in annealing buffer (10 mM Tris-HCl pH 7.4, 10 mM MgCl₂, 50 mM NaCl) by heating at 90°C for 5 min and allowing it to slowly cool to room temperature. The protein-DNA binding reaction was prepared by mixing the reaction buffer (20 mM HEPES pH 7.9, 50mM KCl, 5 mM MgCl₂, 0.5 mM EDTA, pH8.0, 5 mM DTT, 5% glycerol) with the recombinant MyoD (2 μg) and poly [d(I-C)] (Sigma-Aldrich, USA) (0.1 ng) as the nonspecific competitor. The labeled probe was applied to the binding mixture, and the mixture was incubated for 30 min at room temperature. The total EMSA reactions were applied to a 6.5% nondenaturing polyacrylamide gel (0.5X TBE buffer, 6.5% acrylamide/bis-acrylamide (29:1), 6.5% glycerol, 0.1% ammonium persulfate, 0.1%TEMED). After electrophoresis at 100 V for 90 min, the gel was scanned by Biospectrum 500 (Ultra-Violet Products Ltd., USA).

The NF-κB gel shift oligonucleotide (5’-ACTTGAGGGGACTTTCCCAGGGC-3’) (Promega) was radiolabeled using [³²P]-dATP (Amersham Biosciences, Piscataway, NJ, USA) and T4 polynucleotide kinase (GIBCO, Grand Island, NY, USA). The radiolabeled oligonucleotide was separated from unconsumed [³²P]-dATP using a Bio-Rad purification column (Bio-Rad Laboratories, Hercules, CA, USA) eluted with Tris-EDTA buffer (pH 7.5). AGS cell nuclear extracts were incubated at 25 °C for 30 min with the [³²P]-labeled oligonucleotide in buffer containing 12% glycerol, 12 mM HEPES (pH 7.9), 1 mM EDTA, 1 mM DTT, 25 mM KCl, 5 mM MgCl₂, and 0.04 μg/mL poly[d(I-C)] (Sigma-Aldrich, St. Louis, MO, USA). The samples were subjected to electrophoretic separation at 4 °C on a nondenaturing, 5% acrylamide gel. The gel was dried at 80 °C for 2 h after which it was exposed at –80 °C to a radiography film enhanced by an intensifying screen.