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. Author manuscript; available in PMC: 2013 Sep 11.
Published in final edited form as: J Mol Endocrinol. 2004 Dec;33(3):585–608. doi: 10.1677/jme.1.01554

Figure 5.

Figure 5

(A) Mapping of the E-box mediating CLOCK–BMAL1 activation on the Rev-erbα promoters. Trans-activation assays were performed on mutated Rev-erbα promoter fragments in Cos-1 cells. Results are shown in fold activation and are the means±SEM from at least three independent experiments. 40 ng pGL2-luciferase reporter plasmid were used and mixed with 100 ng pCDNA3-Clock and 100 ng pCDNA3-Bmal1 or 200 ng pCDNA3-gal as a neutral vector. (Per1E)3-Luc and (Per1Em)3-Luc are positive and negative controls containing wild-type or mutated E-boxes respectively in front of a minimal promoter. Cells were harvested 48 h after transfection and luciferase activity was analyzed with a luminometer. The E-boxes discussed in the text are shown as black boxes (classical E-box CACGTG) or green boxes (divergent E-box CACATG). An asterisk indicates a mutated E-box. (B) Absolute activities of P1-Luc and a2*P1-Luc (mutated a2) promoters (grey bars) and their CLOCK–BMAL1 activation responses (blue bars) indicated the specificity of the a2 E-box element.