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Measurement of oxidant-induced signal transduction proteins using cell imaging
M E Poynter1, Y M Janssen-Heininger, S Buder-Hoffmann
1Department of Pathology, University of Vermont, Burlington 05405, USA.
Abstract:
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-kappaB). 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.
Insights
Oxidants initiate cell signaling pathways, including mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB (NF-kappaB) cascades. Cell-imaging techniques visualize these oxidant-induced pathways for better understanding of cellular responses.
Area of Science:
- Cellular biology
- Molecular biology
- Biochemistry
Background:
- Oxidants, beyond damaging macromolecules, are crucial initiators of cellular signal transduction.
- Key signaling cascades, such as the mitogen-activated protein kinase (MAPK) pathway (including extracellular-regulated kinases [ERK]) and nuclear factor-kappaB (NF-kappaB) activation, are influenced by oxidants.
- These cascades regulate critical cellular processes including survival, apoptosis, proliferation, transformation, and inflammation.
Purpose of the Study:
- To present protocols for cell-imaging approaches to detect oxidant-induced signaling.
- To highlight the necessity of these techniques for visualizing phosphorylated proteins and nuclear translocation, indicative of pathway activation.
- To discuss the advantages of in situ microscopy-based methods for studying these pathways.
Main Methods:
- Development and application of cell-imaging protocols.
- Utilizing microscopy-based techniques for in situ analysis.
- Complementing traditional biochemical assays.
Main Results:
- Demonstration of effective cell-imaging protocols for visualizing oxidant-induced signaling.
- Successful detection of phosphorylated signaling proteins and their nuclear translocation.
- Validation of microscopy as a key tool for studying these pathways.
Conclusions:
- Cell-imaging approaches are essential complements to biochemical assays for studying oxidant-induced signaling pathways.
- In situ microscopy provides critical insights into the activation of MAPK and NF-kappaB cascades.
- These methods enhance our understanding of how oxidants regulate fundamental cellular functions.