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Optimizing Transfection Conditions for Studying Signal Transduction Pathways

result in a decreased overall transcription rate of the cell, including transcription of the luciferase gene in the reporter plasmid.

In this experiment, we employed two different negative controls: the pFA2-dbd plasmid was used in place of the pFA-ATF2 plasmid and the pcmv-script expression vector without an insert was used in place of the pFC-MEKK plasmid.

Optimizing the PathDetect Cis-Reporting Systems

Since endogenous transcription factors interact with the enhancer elements used in the PathDetect cis-reporting systems, a higher background often results. Optimizing conditions for the experimental and control plasmids or stumuli minimizes such problems. To demonstrate how the cis-reporting systems can be optimized in this situation, we cotransfected the pAP1-Luc reporter vector with either the pFC-MEKK expression plasmid or the empty vector without insert, the pCMV-Script plasmid, into HeLa cells. While holding the concentration of the pAP1-Luc plasmid constant, we varied the amount of the pFC-MEKK plasmid (Figure 2, panel A). Optimized activation of the pAP1-Luc reporting vectors was seen when 10 ng of pFC-MEKK was used.

Figure 2

Sufficient plasmid needs to be available to produce protein to activate signaling pathways that lead to luciferase expression; however, as with the trans-reporting systems, when the transcription activator is too powerful, it can squelch the signal by titrating cellular components necessary for the cellular transcription machinery and other processes. When more than 10 ng of the pFC-MEKK plasmid was used, the system was probably squelched, and activation decreased. The pCM
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