E1 and E2 sites contribute to circadian expression of <i>C/ebpα</i>.

<p>(A) Schematic representation of mutant constructs of <i>C/ebpα</i> promoter region. Either or both E-boxes (sites E1 and E2) were mutated (E1, 5′-CACGTG-3′ to 5′-CACCAC-3′; E2, 5′-CACGTG-3′ to 5′-CACCAC-3′). (B) Real time reporter assay. NIH3T3 cells transfected with indicated mutant constructs were incubated with dexamethasone (100 nM) and then bioluminescence was measured. Plasmid <i>pGL3-dLuc-Cebp-WT</i>, wild type 1.4 kb upstream region of <i>C/ebpα</i> gene;, <i>pGL3-dLuc-Cebp-Mut I</i>, mutant E1 site; <i>pGL3-dLuc-Cebp-Mut II</i>, mutant E2 site; <i>pGL3-dLuc-Cebp-Mut I-II</i>, mutant E1 and E2 sites; <i>mPer2-dLuc</i>, Per2 promoter region (−798 to +331 relative to the cap site) cloned into <i>pGL3-dLuc </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058221#pone.0058221-Ohno1" target="_blank">[23]</a>. Each experiment was repeated 3-5 times and representative data were used for these graphs.</p>