Extraction and Analysis of Transcription Factor-DNA Complexes

Nucleoproteins from HCT116 cells were obtained using the nucleus protein extraction kit supplemented with the protease inhibitor phenylmethanesulfonyl fluoride (PMSF) at a final concentration of 1% (Beyotime, China). Protein quantification was determined using the bicinchoninic acid (BCA) method (Beyotime). A synthetic 5′-end biotin-labelled probe that was consistent with the TF-binding sequence was prepared. The 5′-end biotin-labelled probe, an unlabelled specific competitive probe, and the non-specific competitive mutation probe were generated by GeneCreate Biological Engineering Co (Wuhan, China) as previously described [24 (link)]. The sequences of all probes are shown in Additional file 1: Table S5. Briefly, in each 20 μL reaction, 20 fmol labelled probes were incubated with 2 μg nucleoprotein, together with 4 pmol specific competitive probe or non-specific mutation probe in an ionising environment, and 0.4–0.6 μg anti-CEBPB (Abcam, USA) and anti-TFCP2 (Proteintech, USA) antibodies. Migrating bands representing protein–DNA complexes were developed with a mixture of stable peroxide and luminol/enhancer solutions (LightShift Chemiluminescent EMSA kit, Thermo Fisher). An unlabelled probe at 50 × excess concentration was used as a specific competitor.