Fig. 2: LC3B-AP2 transgenic mouse model facilitates the direct identification of autophagosomal cargoes.

a Generation of mouse model and experimental set-up for proximity labelling. b Western blot of whole splenic cell homogenates from wild-type, heterozygous and homozygous mice. Cells were treated with 10 nM bafilomycin A1 (BafA1) or DMSO for 2 h. These experiments were repeated as biological triplicates (one mouse each) with similar results. c Confocal imaging of biotinylated proteins, AP2 and LAMP1 in proximity-labelled immortalised mouse embryonic fibroblasts (imMEFs) generated from the Lc3b-AP2 mouse model. Light blue arrows indicate co-localisation events with magnifications at the upper-left corners. These experiments were repeated as biological triplicates with similar results. d Electron micrographs of DMSO- or BafA1-treated Lc3b-AP2 imMEFs. Following fixation, cells were incubated with 3,3-diaminobenzidine and H₂O₂ prior to standard embedding and ultrathin sectioning. Squares highlight the autophagosomal structures. Squares that are marked by asterisks are magnified in the upper-right corner. e Homogenates from imMEFs were labelled, then left untreated or incubated with Prot K, Triton-X, or both and followed by immunoblotting for AP2, Biotin, p62 and ATG9A. f Venn diagrams depicting Prot K-protected, biotinylated proteins identified in imMEFs with more than two spectral counts across four biological replicates in both DMSO and BafA1-treated groups. Pearson’s correlation coefficients of the label-free quantity of each protein included in two replicates are displayed. g Volcano plot of proteins from f labelled by LC3B-AP2 in imMEFs. Proteins significantly upregulated in response to BafA1 treatment are highlighted in red (p < 0.05 by unpaired two-tailed Student’s t test, n = 4 biological replicates). Known autophagosomal candidates are labelled with protein names. Source data are provided as a Source Data file.