Please use this identifier to cite or link to this item: https://doi.org/10.4161/auto.28267
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dc.titleDevelopment of a novel method for quantification of autophagic protein degradation by AHA labeling
dc.contributor.authorZhang, J.
dc.contributor.authorWang, J.
dc.contributor.authorNg, S.
dc.contributor.authorLin, Q.
dc.contributor.authorShen, H.-M.
dc.date.accessioned2014-11-26T05:02:48Z
dc.date.available2014-11-26T05:02:48Z
dc.date.issued2014
dc.identifier.citationZhang, J., Wang, J., Ng, S., Lin, Q., Shen, H.-M. (2014). Development of a novel method for quantification of autophagic protein degradation by AHA labeling. Autophagy 10 (5) : 901-912. ScholarBank@NUS Repository. https://doi.org/10.4161/auto.28267
dc.identifier.issn15548635
dc.identifier.urihttp://scholarbank.nus.edu.sg/handle/10635/108907
dc.description.abstractAutophagy is a catabolic process during which cellular components including protein aggregates and organelles are degraded via a lysosome-dependent process to sustain metabolic homeostasis during nutrient or energy deprivation. Measuring the rate of proteolysis of long-lived proteins is a classical assay for measurement of autophagic flux. However, traditional methods, such as a radioisotope labeling assay, are technically tedious and have low sensitivity. Here, we report a novel method for quantification of long-lived protein degradation based on L-azidohomoalanine (AHA) labeling in mouse embryonic fibroblasts (MEFs) and in human cancer cells. AHA is a surrogate for l-methionine, containing a bio-orthogonalazide moiety. When added to cultured cells, AHA is incorporated into proteins during active protein synthesis. After a click reaction between an azide and an alkyne, the azide-containing proteins can be detected with an alkyne-tagged fluorescent dye, coupled with flow cytometry. Induction of autophagy by starvation or mechanistic target of rapamycin (MTOR) inhibitors was able to induce a significant reduction of the fluorescence intensity, consistent with other autophagic markers. Coincidently, inhibition of autophagy by pharmacological agents or by Atg gene deletion abolished the reduction of the fluorescence intensity. Compared with the classical radioisotope pulse-labeling method, we think that our method is sensitive, quantitative, nonradioactive, and easy to perform, and can be applied to both human and animal cell culture systems. © 2014 Landes Bioscience.
dc.description.urihttp://libproxy1.nus.edu.sg/login?url=http://dx.doi.org/10.4161/auto.28267
dc.sourceScopus
dc.subjectAHA
dc.subjectAutophagy
dc.subjectClickchemistry
dc.subjectFlow cytometry
dc.subjectProteolysis
dc.typeArticle
dc.contributor.departmentBIOLOGICAL SCIENCES
dc.contributor.departmentSAW SWEE HOCK SCHOOL OF PUBLIC HEALTH
dc.description.doi10.4161/auto.28267
dc.description.sourcetitleAutophagy
dc.description.volume10
dc.description.issue5
dc.description.page901-912
dc.identifier.isiut000335433700014
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