Extended Data Fig. 2: Evaluation of Atg8 and Atg1 mutants by multiple assays.
From: Recruitment of Atg1 to the phagophore by Atg8 orchestrates autophagy machineries
(A-B) Interaction of Atg8 variants with Atg1 (A), Atg3, Atg4, Atg7, or Atg19 (B) by conventional yeast two-hybrid assay. Mutants in red displayed abnormal interaction. (C-H) Co-immunoprecipitation assay evaluating the interaction between Atg8 variants and Atg3 (C-D), Atg4 (E-F), or Atg7 (G-H). Constructs were expressed under ADH1 promoter in wild-type cells. (C, E, G) Representative immunoblots. (D, F, H) Levels of co-precipitated Atg proteins. Mean ± standard deviation, n = 3 independent repeats. Normalized against sample containing wild-type proteins. Mutants in red displayed abnormal interaction. (I-K) Status of Atg8 lipidation evaluated by immunoblotting. (I) Representative immunoblots. (J, K) Amounts of Atg8-PE, and ratios of Atg8-PE/Atg8. Mean ± standard deviation, n = 3 independent repeats. (L-M) Interaction of Atg1 variants with Atg8 (L), Atg13, Atg17, or Atg11 (M) by conventional yeast two-hybrid assay. (N-S) Co-immunoprecipitation assay evaluating the interaction between Atg1 variants and Atg4 (N-O), Atg13 (P-Q), or Atg17 (R-S). Presented as in (C-H).
