Abstract
Macro-autophagy is a major catabolic process in the cell used to degrade protein aggregates, dysfunctional organelles and intracellular pathogens that would otherwise become toxic. Autophagy also generates energy and metabolites for the cell through recycling of degraded autophagosomal cargo, which can be particularly important for cell viability under stress. The significance of changes in the rates of autophagic flux for cellular function and disease is being increasingly appreciated, and interest in measuring autophagy in different experimental systems is growing accordingly. Here, we describe key methodologies used in the field to measure autophagic flux, including monitoring LC3 processing by western blot, fluorescent cell staining, and flow cytometry, in addition to changes in the levels or posttranslational modifications of other autophagy markers, such as p62/Sqstm1 and the Atg5–Atg12 conjugate. We also describe what cellular stresses may be used to induce autophagy and how to control for changes in the rates of autophagic flux as opposed to inhibition of flux. Finally, we detail available techniques to monitor autophagy in vivo.
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Acknowledgements
This work is supported by funding from NIH RO1 CA162405 (KFM).
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Sharifi, M.N., Mowers, E.E., Drake, L.E., Macleod, K.F. (2015). Measuring Autophagy in Stressed 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_10
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DOI: https://doi.org/10.1007/978-1-4939-2522-3_10
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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