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. 2005 Sep;54(9):1309–1317. doi: 10.1136/gut.2005.065086

Figure 4.

Figure 4

 DNA binding analysis for the cAMP response element binding protein (CREB) II motif. (A) Schematic depiction of the oligonucleotides used for the EMSA experiments. CREB II oligo contains the original sequence found in the putative hepatitis B virus (HBV)-S promoter; CREB II mut has a nonsense mutation in the CREB core recognition site. Competition experiments were performed with non-labelled wild-type (wt) or mutated (mut) CREB consensus (cons) oligonucleotides. (B) In EMSA experiments, nuclear extracts from Huh7 cells (except for lanes 1 and 9) were incubated with radioactive labelled double stranded oligonucleotides corresponding to either the wild-type or a mutated sequence of the upstream CREB (CREB II) motif. In lanes 4–6 and 12–14, competition experiments with increasing concentrations of unlabelled (“cold”) consensus wild-type CREB oligo (molar ratios unlabelled:labelled 10:1, 20:1, 40:1), and in lanes 7–8 and 15–16 with cold consensus mutated CREB oligo were performed. Complex formation (arrows) was detected for the wild-type CREB II oligo, but not with the mutated CREB II oligo, that could be specifically reduced by cold CREB consensus wild-type oligonucleotides but not with mutated CREB consensus oligonucleotide. (C) In supershift EMSA experiments, nuclear extracts from Huh7 cells (except for lane 1) were incubated with radioactive labelled double stranded CREB II oligonucleotide and antibodies specific for CREB1 (lane 4), activating transcription factor (ATF) 1 (lane 5), ATF2 (lane 6), and ATF4 (lane 7). Complex formation (lower arrow) could be supershifted with ATF2 antibody (upper arrow). (D) The same mutation from the CREB II mut oligonucleotide, resulting in a lack of specific CREB complex formation, was introduced into the luciferase reporter constructs, and luciferase activity was measured after transfection in Huh7 cells, as described above. Either the mutation (construct 1B mut) or the deletion (as in construct 2) of the upstream CREB II site resulted in a tremendous decrease in luciferase activity compared with constructs 1 and 1B.