Research Paper Volume 8, Issue 2 pp 245—259

Figure 4. Electrophoresis mobility shift assays (EMSAs) using the activating region. (A) Schematic drawing of EMSA probes. The Hs1, Hs2, and Hs3 probes contain the Har-HK promoter CREB-, c-Myc- and POU-binding regions (bolded), respectively. Three probes were mutated within the CREB- (Ms1), c-Myc- (Ms2) or POU-binding sites (Ms3); mutant regions are bolded. TSS: transcriptional start site; (B) A CRE-binding protein binds Hs1. The Hs1 probe was incubated with 5 μg of brain nuclear extract. BNE, brain nuclear extract; Ns, nonspecific competitor; Smo-CRE, rat somatostatin gene CRE-binding sequence; Ms1, CREB-binding site mutation; (C) c-Myc protein binds Hs2. The Hs2 probe was incubated with 5 μg of brain nuclear extract. ECA39, teratocarcinoma cell ECA39 gene c-Myc-binding sequence; Ms2, c-Myc-binding site mutation. (D) POU protein binds Hs3. The Hs3 probe was incubated with 5 μg of brain nuclear extract. HP: Har-DH-PBAN gene Har-POU-binding site; Ms3: POU-binding site mutation; (E) The in vitro-expressed Har-POU bind Hs3. The Hs3 probe was incubated with 5 μg of brain nuclear extract or 0.1 μl in vitro-expressed Har-POU. HKMBP1, 2, and 3: HK-modulating binding proteins1, 2, and 3, respectively.