To obtain a systems level understanding, experiments should measure the activities of multiple components simultaneously. Such multivariable experiments need to be conducted at multiple levels within the cell. For the systems that we will study in this Center, multivariable experiments need to be conducted at three levels. First, at the level of proteins within the signaling network, we will determine their levels as well as activity state by phosphorylation. For this, we will the reverse protein arrays, wherein cell lysates are spotted onto nitrocellulose coated glass slides and then probed with an array of antibodies (total or phosphospecific) against the components of interest. This approach has been very successful in analyzing cancer systems (1) and we have adapted this for the systems of interest to this Center. This method is powerful because it allows us to use well-characterized antibodies in the range we know is proportional and hence each measurement in this multiplexed assay is in the proportional range. Second, to understand how information processing through the signaling network leads to changes in gene expression it is necessary to determine the activity state of many transcription factors simultaneously. For this, we will use the protein- DNA interaction arrays from Panomics. These arrays allow us to survey the activity state of 340 transcription factors (a list from Panomics which we have cross referenced with Entrez Gene names is attached as Appendix 6) simultaneously. This method has been very useful identifying many transcription factors whose activities are required for neurite outgrowth in response to stimulation by the the cannabinoid CB1 receptor (a Go/i coupled receptor). Third, we will profiles changes in mRNA levels using standard microarray technologies.
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