EMSA Assay for Nuclear Protein Interactions

EMSA were performed essentially as previously describe [44 (link)]. Nuclear proteins were extracted from Beas-2B cells using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (78833, Thermo Fisher Scientific), and then measured using TaKaRa BCA Protein Assay Kit (T9300A, TaKaRa) according to the manufacturer’s instructions. Double-stranded oligonucleotides with or without 5′ biotin-labeled were synthesized (Shanghai Generay Biotech Company, Shanghai, China). The sequences of probes are listed in Additional file 2: Table S3. Electrophoretic mobility shift assay (EMSA) was performed with LightShift™ Chemiluminescent EMSA Kit (20148, Thermo Fisher Scientific) and the anti-p53 antibody (ab1101, Abcam). Briefly, nuclear proteins were pre-incubated with unlabeled probe or anti-p53 antibody in a binding mixture for 10–20 min at room temperature, then incubated with labeled wt-probe or mt-probe for 20 min. The mixtures were electrophoresed in 6% non-denatured polyacrylamide gel, transferred to a nylon membrane (INYC00010, Millipore), and detected biotin-labeled DNA by chemiluminescence.