EMSAs were carried out with double-strand oligonucleotides designed to span the putative transcription factor binding sites (Table 1c). Oligonucleotides were synthesized (Integrated DNA Technologies) with a 5’ 6-FAM label on one of the strands and then annealed to complementary unlabeled oligonucleotides in 10 mM Tris (pH 8.0), 1 mM EDTA (pH 8.0) and 50 mM sodium chloride. Nuclear extracts were prepared from HepG2 cells using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermoscientific Pierce, Waltham, MA), following the manufacturer’s protocol. Protein concentrations were measured by Bio-Rad protein assay (Bio-Rad, Hercules, CA). Protein binding reactions were carried out with 0.2 or 0.4 picomoles of the annealed oligonucleotides and 10 µg of the nuclear extracts in 10 mM Tris-HCl (pH 7.5), 60 mM potassium chloride, 2.5 mM magnesium chloride, 1 mM EDTA, 1 mM DTT, 750 ng of poly (dIdC) and 7% glycerol. Oligonucleotides were incubated with the nuclear extract for 30 min at 25 °C. In competitor assays, unlabeled competitor oligonucleotides in 50-fold molar excess to the labeled oligonucleotides were added to the reaction before addition of the probe. For supershift assays,
