Analysis of the DNA-binding activity of NF-κB p65

Nuclear protein was extracted using a Nuclear Extraction Kit (Active Motif, Carlsbad, CA, USA), following the manufacturer's instructions. A BCA Protein Assay Kit (Pierce; Thermo Fisher Scientific, Inc., Rockford, IL, USA) was used to detect the protein concentration. DNA-binding activity assay for NF-κB p65 in nuclear proteins was determined using a TransAM Assay kit (cat. no. 40096; Active Motif). Briefly, 30 µl binding buffer or binding buffer with 2 µl specific oligonucleotides was added into each well. Subsequently, 20 µl lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) containing nuclear protein was incubated with connection buffer for 1 h at 25°C in the microplates. Subsequently, DNA oligonucleotide-bound protein was detected with anti-p65 (1:1,000) and secondary antibody (1:1,000). Successively, the chromogenic reagent and stop buffer were added into the each hole. Absorbance was detected at 450 nm using a microplate reader (Molecular Devices, Sunnyvale, CA, USA).