The autophagic degradation of Heh1-GFP requires the Atg39 lumenal domain. (A) Western blot (WB) of proteins from whole-cell extracts derived from cells expressing Heh1-GFP in strains with the indicated atg39 alleles cultured in medium lacking nitrogen (SD-N) for 24 h. GFP′ is a stable fragment of GFP in vacuoles. GFP detected with anti-GFP.1 antibody, HRP-conjugated secondary antibodies, and ECL. To assess relative protein loading, a portion of the blot is shown stained with Ponceau S. Position of mol wt standards (in kD) at right. (B) Plot of mean and error bars denoting SD of the percent degradation of Heh1-GFP from three independent experiments as in A. (C and D) WBs of proteins from whole-cell extracts derived from cells expressing the indicated GFP fusions in indicated strains cultured in SD-N medium for 24 h. GFP detected with anti-GFP.1 antibody, HRP-conjugated secondary antibodies, and ECL. To assess relative protein loading, a portion of the blots are shown stained with Ponceau S. Position of mol wt standards (in kD) at right. (E) Plot of mean and error bars denoting SD of the percent degradation of GFP-tagged proteins in the indicated strains from three independent experiments as in C and D. ***, P ≤ 0.001; ****, P ≤ 0.0001 by one-way ANOVA with Tukey’s correction. Source data are available for this figure: SourceData F5.