Figure 4.

Temporal patterns of the site-specific binding of endogenous circadian transcription factors to the Per2 E-box-like sequence. Mouse liver extracts were harvested at 4-h intervals and then subjected to immunoblot analysis with anti-BMAL1 antibody (top panels) or anti-CLOCK antibody (middle panels). Mouse liver extracts were incubated with a double-stranded biotinylated oligonucleotide including the consensus-predicted Per2 E-box-like sequence (CACGTT) or three different Per1 E-boxes (wild-type, WT; mutant, MT), which was immobilized on streptavidin-Sepharose beads. The negative control samples were treated with the beads without an oligonucleotide (No ODN). The resulting precipitates were subjected to immunoblot analysis with anti-BMAL1 antibody (top panels) or anti-CLOCK antibody (middle panels). Temporal expression patterns of the mPer2 and mPer1 genes in mouse liver were assayed by real-time quantitative RT-PCR (bottom panels). Each value represents the average of three independent RT-PCR experiments. The relative levels were normalized to the corresponding Gapdh RNA levels. Peak values of the mPer2 and mPer1 curves were set to 1.