Fig. 4. mAtg9 is present on late endosomes. (A) Western blots of fractions from an endosome preparation gradient from rat liver using antibodies for EEA1 (early endosomes), mAtg9, CI-MPR, TGN38 (TGN), p58 and PDI (ER). To label late endosomes, HRP-biotin was internalized by perfusion for 10 minutes followed by a 20-minute chase before homogenization. HRP-biotin was detected using ExtraAvidin-HRP. Signals were quantified by densitometry, performed using ImageJ. Data are representative of two independent experiments. (B) mAtg9 colocalizes with the CI-MPR in indirect immunofluorescence on HEK293 cells. Inset is enlargement of the peripheral staining in the merge. Bar, 10 µm. (C) Cryoimmunogold labelling of mAtg9 (red arrowheads, 10-nm gold), CI-MPR (green arrowheads, 15-nm gold), on HEK293 cells labelled with 6-nm conjugated BSA-Gold (arrows) internalized for 2 hours. Bar, 200 nm.