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Optimizing Transfection Conditions for Studying Signal Transduction Pathways

for culturing and transfecting cell lines. Because of these variables, it is necessary to optimize experimental conditions to achieve the best results that the PathDetect reporting systems offer. Although many experimental conditions affect the results of PathDetect systems, the amounts of the fusion activator plasmid and expression vector for the gene of interest most dramatically affect the trans-reporting systems. When using the cis-reporting systems, amounts of the reporter plasmid or expression vector can be easily optimized for superior results.

Optimizing PathDetect Trans-Reporting Systems

Figure 1

By varying the amounts of the pFA-ATF2 plasmid, we optimized the conditions for transient transfections of HeLa cells (Figure 1, panel A). The GAL4-ATF2 fusion protein, which is expressed from the pFA-ATF2 plasmid, is strongly activated by the MEKK protein in transient transfection assays.9,10 In this experiment, HeLa cells (1.5 x 105/well) were cotransfected with the indicated amounts of the pFA-ATF2 plasmid, 0.5 g of the pFR-Luc plasmid, and 25 ng of the pFC-MEKK plasmid. The strongest signal for luciferase activity was seen for transfections when 10 ng of the pFA-ATF2 plasmid was used. As the amount of the pFA-ATF2 plasmid further increased, luciferase expression decreased.

When transcription factors are overexpressed, some components of the transcription machinery become depleted; this is described as squelching, and results in decreased transcription.11 One possible explanation for decreased luciferase expression was that the concentration of the GAL4-ATF2 fusion protein e
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