Figure S2.
Assessment of total levels of Atg39 fusion proteins and Atg11 (supplemental to Figs. 2, 3, 5, and 6).(AandB) Western blot (WB) of proteins from whole-cell extracts derived from cells expressing the indicated GFP fusions in indicated strains and drug treatments. GFP detected with anti-GFP.1, 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. In A, the numbers at the bottom of the blot correspond to the relative amount of each protein normalized to GFP-Atg39 using densitometry. (C) Plot of the percentage of cells with zero, one, or two or more NE blebs in GFP-Atg39 and GFP-atg39-(1–312) cells with similar levels of expression (as assessed by comparing total GFP fluorescence of individual cells). At least 100 cells from three independent replicates were evaluated, and mean and SD are plotted. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001 by one-way ANOVA. (D) As in A and B. The numbers at the bottom of the blot correspond to the relative amount of each protein as assessed by densitometry normalized to GFP-Atg39. (E) WB of proteins from whole-cell extracts derived from cells expressing the indicated HA fusions expressed from endogenous ATG39 gene locus after 24 h in SD-N medium. HA detected with anti-HA antibody conjugated to HRP 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. (F) WB of proteins from whole-cell extracts derived from cells expressing GFP-Atg11 alongside the indicated alleles encoding mCherry fusion proteins treated as indicated with rapamycin. GFP detected with anti-GFP.2, 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. (G) As in A and B. Source data are available for this figure: SourceData FS2.

Assessment of total levels of Atg39 fusion proteins and Atg11 (supplemental to Figs. 2 , 3 , 5 , and 6 ). (A and B) Western blot (WB) of proteins from whole-cell extracts derived from cells expressing the indicated GFP fusions in indicated strains and drug treatments. GFP detected with anti-GFP.1, 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. In A, the numbers at the bottom of the blot correspond to the relative amount of each protein normalized to GFP-Atg39 using densitometry. (C) Plot of the percentage of cells with zero, one, or two or more NE blebs in GFP-Atg39 and GFP-atg39-(1–312) cells with similar levels of expression (as assessed by comparing total GFP fluorescence of individual cells). At least 100 cells from three independent replicates were evaluated, and mean and SD are plotted. *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001 by one-way ANOVA. (D) As in A and B. The numbers at the bottom of the blot correspond to the relative amount of each protein as assessed by densitometry normalized to GFP-Atg39. (E) WB of proteins from whole-cell extracts derived from cells expressing the indicated HA fusions expressed from endogenous ATG39 gene locus after 24 h in SD-N medium. HA detected with anti-HA antibody conjugated to HRP 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. (F) WB of proteins from whole-cell extracts derived from cells expressing GFP-Atg11 alongside the indicated alleles encoding mCherry fusion proteins treated as indicated with rapamycin. GFP detected with anti-GFP.2, 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. (G) As in A and B. Source data are available for this figure: SourceData FS2.

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