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

ed luciferase expression indicates that the signal pathway converging at the fusion trans-activator is affected by this compound or stimulus.

As with all transfection experiments, various conditions for experiments using the PathDetect systems should be optimized to achieve the best possible results. Some variables to consider for optimization include the choice of cell line, transfection method, and protocol. In particular, the amounts of the fusion activator plasmid and the expression vector for the gene of interest are the easiest variables to optimize and will have a dramatic effect on transfection results.9

Make New Trans-Activator Plasmids

To offer further research options, Stratagene has designed the pFA-CMV vector15 for use with the trans-reporting systems. Similar to all trans-activator plasmids, the pFA-CMV vector provides expression driven by the CMV promoter### and the capability for G418 selection of stable cell lines. However, the pFA-CMV vector features a multiple cloning site with 10 unique, conveniently arranged restriction sites for insertion of any activation domain sequence. By cloning a transcription factor of interest into the multiple cloning site of the pFA-CMV vector, researchers will have their own fusion trans-activator vectors for studying signaling pathways converging on transcriptional factors of interest.

Specificity of PathDetect Trans-Reporting Systems

A series of experiments (Figure 2) tested the activity and specificity of the trans-reporting systems. For each experiment, the fusion trans-activator plasmid and a known activator were cotransfected with the pFR-Luc reporter plasmid into 1.5 x 105 HeLa cells in 35-mm culture dishes. The total amount of DNA in each reaction was kept constant by adding quantities as needed
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