Measurement of oxidant-induced signal transduction proteins using cell imaging

In addition to their capacity to damage macromolecules, oxidants play impor tant roles in initiation of a number of signal transduction pathways. These include phosphorylation and dephosphorylation of members of the extracellu lar-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-kappa B). These cascades are key to transcription al upregulation of genes important for cell survival, apoptosis, proliferat ion, transformation, and inflammation. To complement biochemical assays, ce ll-imaging approaches are necessary to detect the phosphorylated proteins o f 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 ar e discussed. (C) 1999 Elsevier Science Inc.