EMSA and gel shift assays

Nuclear protein extracts from CRC cells were prepared with nuclear and cytoplasmic extraction reagents (Applygen). EMSA was performed using LightShift Chemiluminescent EMSA Kit (Thermo Scientific) according to the manufacturer's protocol. The 5′-biotin-labelled probe, unlabelled probe and unlabelled mutant probe (Supplementary Table S1) were synthesized in Shanghai Sangon Biological Engineering Technology and Services Company (Shanghai). In supershift assay, anti-E2F1 monoclonal antibody (Merk-Millipore) was pre-incubated with nuclear protein extracts for 5 min at 25°C. The biotin-labelled DNA–protein complex was detected using streptavidin–horseradish peroxidase (HRP) conjugate and LightShift chemiluminescent substrate, viewed in Fusion FX Vilber Lourmat.