Electrophoresis Mobility Shift Assay (EMSA)

This assay utilizes using 2–5 μg of nuclear extract proteins in a total volume of 20 μl containing 20 mM Tris-HCl, pH 7.5, 0.1 M NaCl, 0.35 mM dithiotreitol, 0.5 mM EDTA, 0.5 mM PMSF, 10% glycerol, and 0.5 μl of 2 mg/ml of double-stranded poly(dI-dC) (1 g/reaction). The following oligonucleotide sequences of the probes were used: 1) WT, 5′-TGC GCT CCC GTG CCC CTA GC-3′; 2) 52m, 5′-TGC GCT CCG GTG CCC CTA GC-3′; 3) USF E-box, 5′-TGC GCT CAC GTG CCC CTA GC-3′, where the underlined base indicates the change between wild and 52m in 1) and 2) while the effective E-box for USF in 3). The oligonucleotides were labeled at their 5′ ends with [γ³²P] ATP (NEN Life Science) using T4 polynucleotide kinase. The binding to the double-stranded oligonucleotides was performed by incubating at 4°C for 30 min 10 fmol of ³²P-labeled probes with the previous nuclear extract mix. The DNA–protein complexes were separated by electrophoresis on a 5% polyacrylamide gel and detected by autoradiography of the dried gel. Competitions were performed by addition of 100-fold molar excess of unlabeled double-stranded oligonucleotide competitor to the incubation mixture. Where indicated, the nuclear extracts were preincubated with the specified antibodies (anti-USF1) for 30 min at room temperature and then used for the gel retardation assay. Antibodies against USF1 were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA, USA).