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. 2018 Feb 15;7:e31145. doi: 10.7554/eLife.31145

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© 2018, Gao et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Author response image 2. — (A) Scheme of the purification of autophagosomes from yeast. (B) Total cell lysates from cells grown in YPD medium and starved in SD-N medium for 3 hours. The 15,000 g pellet (P15) fraction from vam3Δ (Atg9-3xFlag, GFP-Atg8) were subjected to density gradient centrifugation and incubated with flag beads to pull down autophagosomes. (C) Detection of Mon1-Ccz1 and Ypt7 on autophagosomes by proteinase K-protection assay. Autophagosomes were collected as described in part B. Equal fractions were then treated with 1 mg/ml proteinase K (PK) in the absence or presence of 1% Triton X-100 (TX).