Detection of NF-κB DNA-binding activity of EpCs and MPs using an electrophoretic mobility shift assay (EMSA)

Following the induction with 2 µg/ml LPS for 6, 12, 24 and 48 h, EpCs and MPs were centrifuged for 5 min with the speed of 500 x g at 4˚C to extract nuclear proteins using NE-PER^™ Nuclear and Cytoplasmic Extraction Reagents (cat. no. 78833; Thermo Fisher Scientific, Inc.). Nuclear extract protein was quantified using a bicinchoninic acid (BCA) assay (Boster Biological Technology). The activity of NF-κB was detected using an EMSA. The following NF-κB oligonucleotide probes were obtained from Beyotime Institute of Biotechnology (cat. no. 1302131410): Forward, 5'-AGTTGAGGGGACTTTCCCAGGC-3' and reverse, 3'-TCAACTCCCCTGAAAGGGTCCG-5'. Binding reactions were performed at room temperature for 20 min in 10 µl binding buffer (cat. no. GS006; Beyotime Institute of Biotechnology), containing 10 µg nuclear extracts, nuclease-free water, EMSA gel-shift buffer and oligonucleotide probes. The biotin-labeled DNA oligo probe without any protein extract was regarded as the negative control. DNA-protein complexes were separated from free DNA probes at 120 V on 5% polyacrylamide gels with 0.5X Tris Boric acid EDTA buffer. Following electrophoresis, the gels were transferred to a nylon membrane and detected using a chemiluminescent substrate (cat. no. 1419701; EMD Millipore). The signal intensity was quantified using an image analyzer (Bio-Rad Laboratories, Inc.).