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. 2009 Aug 31;4(8):e6843. doi: 10.1371/journal.pone.0006843

Figure 7. Analysis of circadian regulation of mouse NTCP promoter.

Figure 7

A. Sequence alignment of conserved DBP binding site in NTCP promoter region of −87 to −108. R = A or G, Y = C or T in the consensus sequence. B. Gel-shift analysis of DBP binding to DBP putative DBP binding site from the NTCP promoter. Assays were performed with radiolabeled probes containing wild type (NTCP) or mutant (NTCP-mut) DBP binding site. The probe containing high affinity DBP binding consensus sequence (PARRE) was used as positive control. For the competition experiment, cold competitors in excess of 5, 10 and 25 folds were added. Protein-DNA complexes and free probes were indicated by arrowheads. C. Luciferase assay of mouse NTCP promoter constructs with the native and mutated, and without the DBP binding site. NTCP84 contains the region from −84 bp to +37 bp (relative to the transcription starting site), NTCP197 encompass the regions of −197 bp to +37 bp and −1 kb to +37 bp respectively. NTCP197mut was generated by changing 3 nucleotides of the putative DBP binding site (from ATTATGCAAC to ATGGGGCAAC) in the NTCP197 reporter.