Abstract
Many experimentally induced or disease-related cellular dysfunctions stress the endoplasmic reticulum, commonly resulting in an accumulation of unfolded proteins in the ER lumen which is sensed by three ER-resident transmembrane proteins, PERK, ATF6, and IRE1. Their activation by such ER stress affects the unfolded protein response, which consists of a shutoff of protein translation and at the same time the switching-on of specific transcription factors that control genes which function to reduce the burden of unfolded proteins to the ER. Here, we describe two sets of methods for monitoring the occurrence of ER stress and UPR signaling in human cells by analyzing markers of activation of all three ER stress sensor proteins. The first set of methods is based on the qualitative and quantitative analysis of UPR-induced transcripts by qPCR. The second set of methods consists of Western blot-based analysis of UPR-induced proteins or protein modifications. Their combined analysis allows assessment of activation of all three ER stress-activated signaling pathways that in combination are characteristic for the UPR.
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Acknowledgments
We would like to thank all the members of our groups, in particular Patricia Cleary for the help with the preparation of figures and Lisa Vincenz who generated Fig. 1b. Figure 2 is an adaptation of a portion of Fig. 4 in Cawley et al., PloS One 2013, 8: e73870. Our work is funded by grants from BELSPO, Belgium, and the Breast Cancer Campaign (2010NovPR13).
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Kennedy, D., Samali, A., Jäger, R. (2015). Methods for Studying ER Stress and UPR Markers in Human Cells. In: Oslowski, C. (eds) Stress Responses. Methods in Molecular Biology, vol 1292. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2522-3_1
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DOI: https://doi.org/10.1007/978-1-4939-2522-3_1
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2521-6
Online ISBN: 978-1-4939-2522-3
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