Electrophoretic mobility shift assay (EMSA) validation of predicted TF-TG interactions

EMSA was carried out using double-stranded Cy5 fluorophore 5′-modified (IDT) DNA probes, in vitro expressed DBDs (see above) and the Gel Shift Assay Core System (Promega). Double-stranded DNA probes were generated by annealing sense and antisense oligonucleotides (see Additional file 1: Table S1) in annealing buffer (10 mM Tris pH 7.5, 1 mM EDTA, 50 mM NaCl) for 3 min at 96 °C, 1 min at 90 °C, 1 min at 85 °C, 3 min at 72 °C, 1 min at 65 °C, 1 min at 57 °C, 1 min at 50 °C, 3 min at 42 °C, and 3 min at 25 °C in a PCR thermocycler. Binding reactions were carried out in a final volume of 9 μl composed of Gel Shift Binding 5x Buffer (20% glycerol, 5 mM MgCl₂, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 50 mM Tris-HCl (pH 7.5), 0.25 mg/ml poly (dI-dC)•poly (dI-dC)); 0.01 μM of Cy5-dsDNA probe covering the motif and flanking region (28 nt); and either 23 ng (RXRB, 10.42 kDa) or 27 ng (NR2C2, 10.73 kDa) of expressed DBD. For EMSA validation with increasing Nr2c2 DBD concentrations, 1× = 27 ng. For kit controls, 0.01 μM of human SP1 DNA probe was combined with 10,000 ng HeLa nuclear extract. Binding reactions were incubated at room temperature for 20 min. Protein-DNA complexes were resolved on 1 mm NuPAGE 4–12% Bis-Tris polyacrylamide gels (Invitrogen) in 0.5× TBE at 100 V for 60 min. Protein-DNA complexes were detected using the Cy5 filter on the ChemiDoc MP (Bio-Rad) system. Exposure settings were adjusted in Image Lab v6.0.1_build34 (Bio-Rad) with same high (5608), low (1152) and gamma (1.0) values set for all associated images.