Selective Accumulation of Aggregation-Prone Proteasome Substrates in Response to Proteotoxic Stress

Florian A. Salomons; Victoria Menéndez-Benito; Claudia Böttcher; Brett A. McCray; J. Paul Taylor; Nico P. Dantuma

doi:10.1128/MCB.01485-08

FIG. 3.

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Increased conjugation of ubiquitin to stress-induced immobile proteins. (A) FRAP curves of GFP-Ub and GFP-Ub^K0,G76V in the nucleus and cytoplasm in untreated and heat-shocked cells. (B) FRAP curves of Luc-GFP and GFP in the nucleus and cytoplasm in untreated and heat-shocked cells. The curves in panels A and B represent the average of 10 cells. (C) Fluorescent micrographs from untreated and heat-shocked cells coexpressing mRFP-Ub and Luc-GFP. The merged images are shown in the lower panels. Scale bar, 10 μm. (D) Western blot analysis of Triton X-100 solubility assays of untreated and heat-shocked parental Mel JuSo cells. Ubiquitin conjugates are detected in total cell lysate (T), detergent-insoluble pellet fraction (P), and soluble fraction (S). (E) Western blot analysis of Triton X-100 solubility assays of untreated and heat-shocked Mel JuSo cells expressing GFP-Ub. GFP-Ub conjugates are detected in total cell lysate (T), detergent-insoluble pellet fraction (P), and soluble fraction (S). α, anti; Mw, molecular weight in thousands.