Two oligonucleotides with 5′-biotin modification representing both the B and D alleles of the Per3 promoter indel were synthesized by MWG Operon (Supplementary Table S1). These oligonucleotides were annealed to form double-stranded probes. Nuclear proteins were isolated from the hippocampus of B6 and D2 mice. EMSA was performed using the Lightshift Chemiluminescent EMSA Kit (Pierce, Rockford, IL, USA). Each 20 μl binding reaction contained 50 fmol of biotin end-labeled target DNA, 1 × binding buffer, 1 μg of Poly(dI.dC) and 25 μg of nuclear protein extract. These reactions were incubated for 20 min at room temperature. In the supershift experiments, 3 μl ATF3 or NRF2 antibody were added, and samples were electrophoresed for 90 min at 100 volts in a 6% polyacrylamide gel (0.5% TBE running buffer). They were then transferred to a Biodyne B membrane (Pierce) and crosslinked in a Stratagene CrossLinker (Stratagene, La Jolla, CA, USA). The Protein–DNA complexes were detected by the ThermoShift Luminescent Nucleic Acid Detection Kit (Pierce). The membranes were exposed with X-ray film.
