Protein extraction and electrophoretic mobility shift assay (EMSA)

A Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime; Shanghai) and quantified proteins from a BCA Protein Assay Kit (Beyotime; Shanghai) were used for extraction and concentration determination. Protocols followed the manufacturer’s instructions. The procedure for EMSA was described in the instructions for a LightShift^® Chemiluminescent EMSA Kit (Thermo Scientific; USA). In brief, a control Epstein-Barr nuclear antigen (EBNA) system included three reactions: specific binding reactions, competition reactions and negative reactions that contained no protein extract, while the test system included additional TFBS mutation reactions. The biotin end-labeled target DNA, unlabeled DNA and TFBS mutation DNA were amplified by PCR. The unlabeled DNA sequences were the same as the biotin-labeled sequences, and primers of the oligos for biotin-labeled target DNA and TFBS mutation DNA are listed in Table S1. Next, 0.1 µM biotin-labeled target DNA (2 µl), 10x binding buffer (2 µl), 1 µg/µl poly (dI-dC) (1 µl), 50% glycerol (1 µl), 1% NP-40 (1 µl) and 100 mM MgCl2 (1 µl) were in each reaction, and 10 µM unlabeled DNA (4 µl) was in a competition reaction while 10 µM TFBS mutated DNA (4 µl) was in a TFBS mutation reaction. All reactions contained 10 µg of protein extract except the negative control. After incubation at room temperature for 30 min, 20 µl of each binding sample was loaded onto a 6% polyacrylamide gel. A 0.45 µm nylon membrane was used for electrophoretic transfer on ice with 0.5X TBE at 380 mA (∼100 V) for 30 min. Biotin-labeled DNA was detected by chemiluminescence after the membrane was blocked for 15 min and washed 5 times (5 min/wash).