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. 2019;619:319-336.
doi: 10.1016/bs.mie.2018.12.034. Epub 2019 Feb 4.

Western Blot Analysis of the Autophagosomal Membrane Protein LGG-1/LC3 in Caenorhabditis Elegans

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Western Blot Analysis of the Autophagosomal Membrane Protein LGG-1/LC3 in Caenorhabditis Elegans

Alexander Springhorn et al. Methods Enzymol. .

Abstract

Autophagy is being studied intensively in Caenorhabditis elegans in the context of protein homeostasis and aging. However, in contrast to the yeast and mammalian autophagosomal membrane proteins Atg8 and LC3, lipidation of the C. elegans ortholog LGG-1 with phosphatidylethanolamine has rarely been investigated by western blotting. We attribute this shortcoming to technical problems with separating the nonlipidated from the lipidated LGG-1 protein by gel electrophoresis. Our new protocol for Western blot analysis is applicable for both the detection of transgenic and endogenous LGG-1 proteins and provides a quantifiable method to assess autophagic flux. As a proof of principle, we use this assay to analyze the role of the transcriptional master regulator HLH-30/TFEB in starvation-induced autophagy.

Keywords: ATG-18; ATG-4.1; Atg8/LC3; Autophagic flux; Autophagy; C. elegans; HLH-30; LGG-1; SQST-1; Starvation; Ubiquitin; Western blot; p62.

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