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GFP-Atg8 processing assay. WT cells expressing GFP-Atg8 from ATG8 endogenous promoter (Y1408) were grown to exponential phase in sulfur free (SF)-medium supplemented with 0.1 mM Met and starved as described in Methods. Cells were collected before (t0), and 2, 4, 6 and 8 h after starvation. Protein extracts were resolved by SDS-polyacrylamide gel electrophoresis and analyzed by western blot using antibodies against GFP and Pgk1 (loading control). Molecular weight markers are in kDa. Quantification was performed as described in Methods. GFP-Atg8 expression and GFP release are relative to the WT at 8 h. Data are mean of two independent cultures.

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