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Assess the In Vivo Activation of Signal Transduction Pathways with,,,PathDetect Reporting Systems

includes the following plasmids: a pathway-specific fusion trans-activator plasmid, which is an in-frame fusion of the activation domain of a specific transcription factor, and the DNA binding domain of the yeast transcriptional activator GAL46,7; the pFR-Luc reporter plasmid for expression of the Photinus pyralis (firefly) luciferase8 gene controlled by a synthetic promoter that contains the yeast GAL4 binding sites; a positive control plasmid that constitutively expresses an activated kinase that is known to activate the trans-activator protein; and a negative control plasmid that contains only the DNA binding domain of the yeast GAL4.

The PathDetect trans-reporting systems are easy to use.5 When the pFR-Luc plasmid is transfected into mammalian cells alone or together with the pFC2-dbd plasmid, which contains only the GAL4 DNA binding domain, little or no luciferase is expressed, establishing the background level of luciferase expression for the cell line used. When a fusion trans-activator plasmid, the pFR-Luc reporter plasmid and a plasmid encoding an uncharacterized gene are cotransfected into mammalian cells, either direct or indirect phosphorylation of the fusion trans-activator protein by the uncharacterized gene product will cause increased transcription of the luciferase gene from the reporter plasmid. Thus, increased luciferase activity indicates that the uncharacterized gene product is involved in a specific pathway. In contrast, if expression of the gene of interest neither directly nor indirectly results in the phosphorylation of the activator fusion protein, luciferase expression will not significantly exceed the background level. Similarly, if cells transfected with pFR-Luc and a fusion trans-activator plasmid are treated with a compound or other extracellular stimulus, increas
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