Nuclear extracts from HB1.F3 cells were prepared as described earlier (Kim et al., 2013 (link)). Sense and antisense oligonucleotides were annealed and then end-labeled with [γ-32P]ATP (Amersham) and T4 polynucleotide kinase. Labeled probes were purified on 19% non-denaturing polyacrylamide gels. The DNA–protein binding reaction was performed in a final volume of 20 μl reaction buffer containing 10 mM Tris (pH 7.6), 50 mM NaCl, 0.5 mM dithiothreitol, 0.5 mM EDTA, 1 mM MgCl2, 5% glycerol, and 250 μg of poly(dI–dC) per milliliter. Nuclear extract (20 μg of protein) was added to the reaction buffer in the absence or presence of unlabeled competitor DNA and pre-incubated for 15 min on ice. Radioisotope-labeled probes (50,000 cpm) were added, and the mixture was incubated for a further 30 min at room temperature. To resolve DNA–protein complexes, electrophoresis was performed on a 5% non-denaturing polyacrylamide gel. Gels were fixed, dried, and visualized by autoradiography. The oligonucleotides employed were as follows (only sense strands presented):
NMU-NurRE, 5'-GTTCCTCACCTTTCAAAGGGAGGTCAAATA-3';
NMU-mtNurRE, 5'-GTTCCCTGTT TTTCAAAAACAG GTCAAATA-3'; and
G0S2-NBRE2, 5'-CATCACTGACCTTTGCAATT-3'.
NMU-NurRE, 5'-GTTCCTCACCTTTCAAAGGGAGGTCAAATA-3';
NMU-mtNurRE, 5'-GTTCC
G0S2-NBRE2, 5'-CATCACTGACCTTTGCAATT-3'.
