Misfolded GFP-DD_FKBP and Ub conjugates are stable at IBs. (A) Misfolded GFP-DD_FKBP is stable at IBs. U2-DD^FKQ91 cells were treated with 1 µg/ml dox and 1 µM shield-1 for 48 h and imaged by time-lapse microscopy after washout of shield-1. (B) Quantification of mean GFP-DD_FKBP fluorescence at IBs in the cytosol/nucleus of cells with IBs present and in the cytosol/nucleus of cells without IBs in cells (n > 10) imaged in A. (C) Misfolded GFP-DD_FKBP is sequestered at IBs. U2-DD^FKQ91 cells were treated with 1 µg/ml dox for 48 h. Cells were subsequently imaged by time-lapse microscopy after addition of 1 µM shield-1 and 25 µM emetine to prevent accumulation of cytosolic and nuclear GFP-DD_FKBP fluorescence (for addition of emetine only; Fig. S3 A). Arrowheads indicate IBs that formed after addition of shield-1, and arrows indicate IBs that were present before addition of shield-1. (D) Quantification of mean GFP-DD_FKBP fluorescence at IBs and in the cytosol/nucleus in cells (n > 10) imaged in C. (E) YFP-Ub accumulates at IBs. U2-Ub-Q91 cells were treated with 1 µg/ml dox for 48 h and imaged by time-lapse microscopy. (F) Quantification of YFP-Ub and httQ91-mCherry fluorescence at IBs in cells (n > 10) imaged in E. IB start indicates the frame in which httQ91-mCherry IBs were first detected. (G) Ub conjugates are stable at IBs. U2-Ub-Q91 cells were treated with 1 µg/ml dox for 48 h. Cells were subsequently treated for 3 h with 10 µM E1 inhibitor and imaged by time-lapse microscopy. (H) Quantification of YFP-Ub fluorescence at IBs in cells (n > 15) imaged in G. Arrowheads indicate IBs in A, E, and G. Time stamps indicate elapsed time in minutes. Bars, 10 µm. Data points indicate mean ± SD. Statistically relevant differences (α = 0.05): **, P < 0.0001.