Ab2743

Supershift assays were carried out using the Dig Gel Shift Kit 2nd Generation (Roche). Nuclear proteins from tilapia liver (10 µg) were obtained as previously described (García-G et al. 2007 (link)) and incubated on ice for 30 min with 2 μg of Jab1 antibody (Abcam, ab124720) and/or 8 μg of TR antibody (Abcam, ab2743) in binding buffer (1 × 10 -5 M Tris-HCl pH 8.0, 3 × 10 -4 M KCl, 10 (v/v) glycerol, 0.2 × 10 -3 M MgCl 2 , 0.5 μg/μL BSA and 1 μg/μL poly dI-dC). A DIG-labeled Direct Repeat 4 response element (DR4, 5′-AGC TTC AGT CAC AGG AGG TCA GAG AG-3′) 232:3 was added, and the binding reactions were incubated for 15 min on ice followed by a 15-min incubation at room temperature. The reaction was loaded onto a 6.5% native polyacrylamide gel and resolved at 120 V over the course of 2 h. The DNA-protein complexes were visualized by following the instructions of the manufacturer. Excess cold DR4 or chicken β-actin oligonucleotides (5′-CTG GGA TGA TAT GGA GAA GAT CTG GCA CC-3′) were added to the binding reaction to evaluate specific and non-specific binding, respectively.