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|Title:||Measuring signaling by the unfolded protein response|
|Citation:||Methods in Enzymology - Volume 491 - The Unfolded Protein Response and Cellular Stress, Part C, 2011 / Conn, P. (ed./s), pp.261-292|
|Publisher Place:||United States|
|Series/Report no.:||Methods in Enzymology|
|David J. Cox, Natalie Strudwick, Ahmed A. Ali, Adrienne W. Paton, James C. Paton, and Martin Schröder|
|Abstract:||The unfolded protein response (UPR) is activated by accumulation of unfolded proteins in the endoplasmic reticulum (ER). The unfolded protein response is associated with many diseases, including cancer, metabolic diseases such as type II diabetes and fatty liver diseases, and neurodegenerative diseases, for example, Alzheimer's disease. The UPR is also activated by numerous toxic chemicals and modulates drug action. Therefore, the UPR becomes increasingly important in toxicological and pharmacological research. In mammals, the UPR is transduced through three parallel signaling pathways originating at the ER-resident transmembrane protein kinase-endoribonucleases (RNase) IRE1, the protein kinase PERK, and a family of type II transmembrane transcription factors, whose most prominent member is ATF6α. We discuss methods to experimentally activate the UPR in the yeast Saccharomyces cerevisiae and in cultured mammalian cells. We summarize methods to monitor activation of the three arms of the UPR, while providing detailed protocols for select, reliable assays. To monitor activation of the IRE1 branch, a Northern blotting protocol to monitor splicing of HAC1 mRNA in yeast and a reverse transcriptase-PCR assay for processing of the IRE1 RNase substrate XBP1 in mammalian cells are presented. Activation of the IRE1 kinase activity can be assayed by immunoblotting for IRE1 autophosphorylation. Activation of the PERK branch is monitored via phosphorylation of the translation initiation factor eIF2α, induction of CHOP at the mRNA and protein level, and induction of ATF4 at the protein level. Activation of ATF6 is assayed in Western blots through the appearance of its processed 50 kDa soluble cytosolic fragment. We summarize reverse transcriptase-PCR protocols to measure activation of target genes selectively induced by the three branches of the UPR and histological assays for UPR activation in tissue sections. This repertoire of methods will enable the newcomer to the UPR field to comprehensively assess the activation status of the UPR.|
|Keywords:||ATF6; eIF2α; Endoplasmic reticulum; HAC1; IRE1; PERK; Stress; Translational attenuation; Unfolded protein response; XBP1|
|Rights:||Copyright © 2011 Elsevier Inc. All rights reserved.|
|Appears in Collections:||Molecular and Biomedical Science publications|
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