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Figure 4 | BMC Molecular Biology

Figure 4

From: cAMP response element binding protein (CREB) activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

Figure 4

Schematic representation and biological activity of the nuclear targeted mutant of the catalytic subunit of cAMP-dependent protein kinase. (A) Schematic representation of the modular structure of the catalytic subunit of cAMP-dependent protein kinase (Cα) and the mutant NLSCα. The wild-type enzyme is myristylated as indicated (Myr). The sequence of the nuclear localization signal derived from the SV40 large T antigen and the triple FLAG epitope in NLSCα are shown. (B) Western Blot analysis of HepG2 cells transfected with an expression vector encoding NLSCα. As a control, extracts from mock transfected HepG2 cells were analyzed. Western blots were probed with an antibody against the FLAG-tag. (C) Modular structure of the GAL4-CREB fusion protein. The protein consists of the DNA-binding domain of GAL4 (amino acids 1–147) and the activation domain of CREB (amino acids 1–281). The reporter plasmid pUAS5luc contains a transcription unit encompassing the luciferase open reading frame and a minimal promoter that consists of five copies of the upstream activating sequence (UAS), a TATA box derived from the HIV long terminal repeat and the initiator element from the adenovirus major late promoter. The reporter plasmid pUAS5luc (1 μg/plate) was transfected into HepG2 cells together with the pRSVβ internal standard plasmid (2 μg/plate) and either an expression vector encoding the DNA binding domain of GAL4 (plasmid pM1) or the GAL4-CREB fusion protein (1 μg plasmid/plate). In addition, cells were transfected with an expression vector encoding either the wild-type (Cα) or mutated (NLSCα) form of cAMP-dependent protein kinase (100 ng/plate). Forty-eight hours post-transfection cell extracts were prepared and the β-galactosidase and luciferase activities of these extracts were determined.

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