2.12. Electrophoretic Mobility Shift Assays (EMSA)

Nuclear proteins were prepared as described previously [22]. EMSAs were performed by using a Lightshift chemiluminescent EMSA kit according manufacturer’s instruction (Thermoscientific, IL, USA). The biotin-upper and biotin-lower strand of Stat3 probe (5′-cttcatttcccggaaatcccta-Biotin-3′ and 5′-tagggatttccgggaaatgaag-Biotin3′) were used. The biotin-labeled DNA probes were incubated with PAA-treated nuclear proteins in a final volume of 20 μL EMSA buffer containing 1 μg/μL poly (dI-dC) at room temperature for 20 min. The protein/DNA complexes were run on a 4% polyacrylamide nondenaturing gel in 0.5× TBE (45 mM tris-borate and 1 mM EDTA) and detected using a chemiluminescent nucleic acid detection kit (Thermo Scientific).