The growth and survival of cancer cells are often driven by constitutive activity in the mitogen-activated protein kinase (MAPK) and phospho-inositide 3-kinase (PI3K)/AKT signaling pathways. Activity in these signal transduction cascades is known to contribute to the uncontrolled growth and resistance to apoptosis that characterizes tumor progression. There is now a great deal of interest in therapeutically targeting these pathways in cancer using small molecule inhibitors. In this chapter, we describe methods to measure constitutive MAPK and AKT activity in melanoma cell lines, with a focus upon Western blotting, phospho-flow cytometry, and immunofluorescence staining techniques.
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