Figure 7. Per1 interacts with an E-box response element in the αENaC promoter.

A. Circadian clock proteins typically bind E-box elements. The αENaC promoter was analyzed for E-box elements using TFSEARCH and TESS. Four putative elements were found and are designated by the vertical gray lines. The position of each E-box element relative to the transcription start site is indicated. Complete DAPA probe sequences are listed in Methods. The consensus E-box sequence contained within each probe is: E-box 1, CAGCTG; E-box 2, CAGAGG; E-box 3, CAGGTG; E-box 4, CAGGTG. B. DNA affinity purification assays (DAPA) were performed using nuclear extract from duplicate mpkCCD_c14 cell samples. Biotinylated probes representing each putative E-box from the αENaC promoter were incubated with nuclear extract samples and pulled down using streptavidin agarose beads. Samples were analyzed using Western blot analysis with an anti-Per1 antibody (Affinity BioReagents) or an anti-Clock antibody (Thermo Fisher Scientific). Data are representative of three independent experiments. In the lower panel, separate Western blot analysis for actin was performed on 20 μg of the input nuclear extract samples used in the DAPA experiment. C. DAPA experiments were performed as described for Panel B, but using only E-box 3 incubated with nuclear extract from non-target or Per1–8 siRNA transfected mpkCCD_c14 cells. In the bottom panel, separate Western blot analysis for actin was performed on 20 μg of the input nuclear extract samples used in the DAPA experiment.