Electrophoresis mobility shift assay (EMSA)

Nuclears extracts from cells were performed with EMSA Detection Kit (Key-GEN, Nanjing, China) according to the manufacturer’s instructions. The NF-κB oligonucleotide comprised the sequence: 5’-AGCTATGTGGGTTTTCCCATGAGC-3’. To confirm the specificity of NF-κB, a 50-fold excess of NF-κB oligonucleotide, which was unlabeled, was added to the reaction mixture as a competitor. For EMSA, proteins were incubated with a NF-κB-specific ³²P-labeled oligonucleotide and mix for binding. For supershift assay, antibodies were preincubated to the sample of interest prior to incubation with radiolabeled probe. The complexes formed were analyzed using Phosphor Imager Technology.