Conditionally destabilized reporters are recruited to IBs. (A) GFP-DD_FKBP is stabilized by shield-1. U2OS cells stably expressing GFP-DD_FKBP and tetracycline-inducible httQ91-mCherry (U2-DD^FKQ91) were treated with 1 µM shield-1 or subjected to shield-1 washout and imaged by time-lapse microscopy. (B) GFP-DD_FKBP undergoes Ub-dependent degradation. GFP-DD_FKBP fluorescence in U2-DD^FKQ91 cells was determined by flow cytometry analysis in the presence of 1 µM shield-1 (S1) or after washout of shield-1 for 6 h in the presence or absence of 10 µM E1 inhibitor. (C) U2-DD^FKQ91 cells were treated with 1 µM shield-1 (top) or 1 µg/ml dox (bottom) for 72 h and analyzed by flow cytometry. (D–F) Misfolded but not folded GFP-DD_FKBP is recruited to httQ91-mCherry IBs. U2-DD^FKQ91 cells were treated with 1 µg/ml dox for 48 h and imaged by time-lapse microscopy in the absence of shield-1 (D), in the presence of 1 µM shield-1 (E), or after shield-1 washout (F). (G) Cells stably expressing DD_DHFR-GFP and tetracycline-inducible httQ91-mCherry (U2-DD^DHQ91) were treated with 10 µM TMP (top) or 1 µg/ml dox (bottom) for 72 h and analyzed by flow cytometry. (H and I) Misfolded but not folded DD_DHFR-GFP is recruited to httQ91-mCherry IBs. U2-DD^DHQ91 cells were treated with 1 µg/ml dox for 48 h and imaged by time-lapse microscopy in the presence of 10 µM TMP (H) or after TMP washout (I). Time stamps indicate elapsed time in minutes. For montages of httQ91-mCherry, t = 0 min indicates the frame in which IBs were first observed, and negative values correspond to time in minutes preceding this frame. (A, D–F, H, and I) Microscopy panels are representative of n > 50 (A and D–F) and n > 10 (H and I) independent observations. Arrowheads indicate IBs. (B, C, and G) For flow cytometry, n > 10,000 cells were analyzed. Ctl, control. Bars, 10 µm.