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Optimizing Transfection Conditions for Studying Signal Transduction Pathways

xceeded the level able to be phosphorylated by a limited amount of upstream activators in the cells. Phosphorylated and nonphosphorylated species of the GAL4-ATF2 fusion compete for the GAL4 binding sites on the reporter plasmid. Because transcription of the luciferase gene on the reporter plasmid is activated only by the phosphorylated GAL4-ATF2 fusion protein, lower levels of luciferase activity may occur.

As a negative control, we varied the amounts of the pFA2-dbd plasmidwhich does not contain an activation sequencein parallel transfections in place of the pFA-ATF2 trans-activator plasmid. As expected, this control showed very low background luciferase expression.

Transfecting varying amounts of the expression plasmid containing the gene of interest also had dramatic effects. Hence, another way to optimize induction of the PathDetect system is by transfecting varying amounts of the expression plasmid while maintaining constant amounts of the trans-activator plasmid and reporter plasmid.

In Figure 1, panel B, HeLa cells (1.5 x 105/well) were cotransfected with 10 ng of the pFA-ATF2 plasmid, 0.5 g of the pFR-Luc plasmid, and varying amounts of the pFC-MEKK plasmid. Maximum activation of luciferase expression was seen when 10 ng of the pFC-MEKK plasmid was used per transfection. In this experiment, when the amount of the pFC-MEKK plasmid increased above 10 ng, luciferase activity declined. As before, the GAL4-ATF2 fusion protein is phosphorylated by overexpressing the MEKK protein and binds to the GAL4 binding domain. However, in this scenario, too much of the MEKK protein could cause other signal transduction pathways to overactivate, which would deplete some of the common factors (e.g., coactivators) in the cells transcription machinery. When factors are depleted, they
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