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. 2011 Jan 21;12(2):149–156. doi: 10.1038/embor.2010.203

Figure 3.

Figure 3

Aggresome-targeted proteins are degraded by autophagy. (A) Analysis of SODG85R-GFP (green) localization with aggresome marker vimentin, the autophagic receptors p62 and NBR1, the autophagosome marker LC3 and the lysosomal marker LAMP2 (all in red) in BAG3/Hsp70-transfected HEK cells. Representative merged images of cells with cytoplasmic, pre-aggresomal and aggresomal SOD distribution are shown. Arrows indicate LC3-positive autophagosomes. Scale bar, 5 μm. (B) Transmission electron microscope analysis of SODG85R-GFP aggresome in cells transfected as in (A). Marked area in (a) is shown magnified in (b). Arrows, autophagosomes. Arrowheads, autolysosomes. Scale bar, 1 μm. (C) HEK cells transfected for 8 h with SODG85R-GFP along with FLAG-BAG3 and HA-Hsp70, as indicated, were treated for 24 h with BafA1 (0.1 μM). Indicated proteins were analysed by immunoblotting. Diagram shows the ratio of SODG85R-GFP levels in BafA1-treated cells to those in untreated cells. (D) Same analysis as in (C) but with SODWT-GFP. (E) Similar analysis as in (C) but in cells transfected 24 h before with nonsense or atg7 siRNA. (F) HEK cells were transfected with SODG85R-GFP and indicated FLAG-tagged BAG3 constructs together with HA-Hsp70, and lysosomal degradation of SODG85R-GFP was examined as in (C). Corresponding immunoblots are shown in supplementary Fig S3F online. Ag, aggresome; BafA1, bafilomycin A1; GFP, green fluorescent protein; HA, haemagglutinin; Hsp70, heat-shock protein 70; N, nucleus; NS, not significant; nons, nonsense; PxxP, proline-rich repeat; siRNA, short interfering RNA; SOD, superoxide dismutase; SODWT, wild-type SOD.