Electrophoretic mobility shift assay

CBF3 cDNA was cloned into pET-28a vector using primers CBF3-HisF and CBF3-HisR (Table S1). Soluble CBF3-His protein was expressed in Escherich coli strain BL21 and purified using HisPur™ Cobalt Resin (Thermo, USA). The 5′ biotin-labeled double-stranded oligonucleotides GA2ox7-L2 was used as a probe while non-labeled GA2ox7-L2 and GA2ox7-L2-m (mutated) were used as competitors, respectively. The labeled probes (3 pM) with or without unlabeled competitors were incubated for 20 min at room temperature with 4 μg of purified CBF3-His fusion protein in binding buffer (25 mM HEPES-KOH buffer at pH 7.9, containing 50 mM KCl, 0.5 mM EDTA, 0.5 mM DTT and 10% glycerol) supplemented with 20 pM poly (dI-dC). The resulting DNA-protein complexes were resolved by electrophoresis on a 6% non-denaturing polyacrylamide gel in 0.5 × TBE buffer and transferred by electroblotting to PVDF membranes. After crosslinked under UV (120 mJ/cm², 254 nm), the signal was visualized according to manufacturer instruction of LightShift Chemiluminescent EMSA kit (Thermo, USA).