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. Author manuscript; available in PMC: 2023 Dec 7.
Published in final edited form as: Nat Cell Biol. 2016 Jun 13;18(7):765–776. doi: 10.1038/ncb3372

Figure 7.

Figure 7

Secretion of misfolded proteins does not involve autophagosomes or exosomes. (a) Two established UPS pathways that involve an intracellular vesicle carrier. (b) GFP1–10 is not secreted by exosomes. Conditioned media (16h) collected from cells transfected with GFP1–10 together with the indicated USP19 variants were subjected to differential centrifugation and immunoblot analysis. S, supernatant; P, pellet. The faint bands in the outlined areas are caused by serum proteins crossreacting with the antibodies. (c,d) Most MAPS vesicles are not autophagosomes. COS7 cells transfected with mCh–GFP1–10 and mCe–USP19 were photobleached, methanol-fixed, and stained with LC3 antibodies. Arrowheads in c,d show examples of MAPS vesicles not labelled by LC3 antibodies. Scale bar, 5 μm. (d) A 3D reconstructed image after z-section confocal analysis. (e) Secretion of Ubl4A–FLAG is not affected by starvation. Cells transfected with the indicated plasmids were either incubated in complete medium or an EBSS starvation medium. Media collected at the indicated time points and lysates prepared at the end of the chase were analysed by immunoblotting. (f,g) GFP1–10 secretion does not require GRASPs. (f) Secretion of GFP1–10 was analysed in cells transfected with GFP1–10 and FLAG–USP19 together with the indicated siRNAs. Knockdown of GRASP65 was confirmed by immunoblotting. (h) The same as in g, except that GRASP55 and Tsg101 were included and that gene knockdown was confirmed by quantitative PCR with reverse transcription (indicated in green labels). Unprocessed original scans of blots are shown in Supplementary Fig. 8.