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In Vivo Signal Transduction Pathway Reporting Systems

nd.

The reporter vector, the pFR-Luc plasmid (figure 1, panel B), is identical in all three PathDetect kits described here and consists of the firefly luciferase gene14 controlled by five repeats of GAL4 binding element followed by a basic transcriptional promoter (TATATA). When pFR-Luc is cotransfected into mammalian cells alone or in the presence of the PathDetect fusion activator plasmid, little or no luciferase is expressed. This level of luciferase activity will constitute the background expression level for the cell line used. When the PathDetect activator and reporter vectors are cotransfected into mammalian cells along with an uncharacterized gene, luciferase activity will either remain at the background level or will increase. If expression of the gene results in direct or indirect phosphorylation of the activator fusion protein, the luciferase gene will be expressed, which is a strong indication that the gene of interest is involved in the pathway being evaluated. 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.

Specificity of PathDetect Systems

figure 3

A series of experiments (figure 3) tested the level of activity and the specificity of the PathDetect systems. For
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