Measurement of oxidant-induced signal transduction proteins using cell imaging

In addition to their capacity to damage macromolecules, oxidants play important roles in initiation of a number of signal transduction pathways. These include phosphorylation and dephosphorylation of members of the extracellular-regulated kinase (ERK) family of the mitogen-activated protein kinase (MAPK) cascade and events leading to activation of the transcription factor nuclear factor-kappaB (NF-κB). These cascades are key to transcriptional upregulation of genes important for cell survival, apoptosis, proliferation, transformation, and inflammation. To complement biochemical assays, cell-imaging approaches are necessary to detect the phosphorylated proteins of these cascades and their nuclear translocation, i.e., activation in cells. Protocols for these studies are presented, and the advantages of in situ microscopy-based techniques to detect oxidant-induced signaling pathways are discussed.