DNA pull-down assay was performed as described (Ogawa et al. 2014 (link)). A 74-bp DNA fragment containing the E2F1 binding site (-24/+50) (Fig. 2B) was amplified by PCR using biotinylated primers. 30 µl nuclear extract (100 µg) was added to 180 µl of 5 mM Tris (pH 8.0)-14% glycerol buffer and pre-incubated on ice. 15 µl of poly(dI-dC)(dI-dC) (7.5 µg) and 18 µl of 5 x binding buffer (300 mM KCl, 60 mM HEPES [pH 7.9], 20 mM Tris-HCl [pH 8], 0.5 mM EDTA, 25% glycerol and 5mM DTT) and 6 µl of probe (1 µg) were incubated at room temperature for 15 min. DNA–protein complexes were collected with 10 µl Dynabeads M-280 Streptavidin magnetic beads (Life Technologies) and analyzed by immunoblotting.