CLOCK binds to E1 and E2 sites in living cell.

<p>(A) Chromatin immunoprecipitation (ChIP) assay for CLOCK and E-boxes of <i>C/ebpα</i> promoter. E-box sites are shown as ovals on the schematic. Horizontal bars indicate amplified regions (E-box unrelated sequence, from −2239 to −2063; E1 site, from −1240 to −1128; E2 site, from −295 to −137.) NIH 3T3 cells were rapidly sampled and processed every 6 hours after dexamethasone stimulation. Chromatin immunoprecipitation was performed using antibodies against CLOCK, and enrichment of E-boxes containing sequence of the promoter region of <i>C/ebpα</i> were measured using quantitative PCR. Data were normalized to a control condition (Normal Rabbit IgG). The ChIP assay was repeated two times. Each time point comes from triplicate experiments. Data are presented as mean ± SEM (n = 3, *: p<0.0001). (B) Temporal mRNA expression of endogenous <i>C/ebpα</i> in NIH 3T3.Total RNA was extracted every 6 hours after dexamethasone stimulation and mRNA transcripts were quantitated relative to <i>Rplpo</i> transcript expression (housekeeping gene) by using quantitative RT-PCR. Each time point comes from triplicate experiments. Data are presented as mean ± SEM (n = 3, *: p<0.05).