Misfolded Fluc-GFP is recruited to IBs. (A and B) smFluc-GFP and wtFluc-GFP are recruited to IBs and distinct foci. Cells were transiently transfected with htt91-mCherry and smFluc-GFP (A) or wtFluc-GFP (B) for 48 h and imaged by time-lapse microscopy. Arrowheads indicate IBs, and arrows indicate Fluc-GFP foci that do not colocalize with IBs. Time stamps indicate elapsed time in minutes. t = 0 min indicates the frame in which IBs were first observed, and negative values correspond to time in minutes preceding this frame. Bars, 10 µm. (C) GFP-DD_FKBP decreases the concentration at which httQ91-mCherry forms IBs. Cells stably expressing tetracycline-inducible httQ91-mCherry (U2-Q91) were transiently transfected with GFP-DD_FKBP in the presence of vehicle or 1 µM shield-1 for 24 h. After addition of 1 µg/ml dox for 72 h, cells expressing low or high levels of GFP-DD_FKBP were analyzed by PulSA. (D) DD_DHFR-GFP decreases the concentration at which httQ91-mCherry forms IBs. U2-Q91 were transiently transfected with DD_DHFR-GFP in the presence of vehicle or 10 µM TMP for 24 h. After addition of 1 µg/ml dox for 72 h, cells expressing low or high levels of DD_DHFR-GFP were analyzed by PulSA. (E) wtFluc-GFP decreases the concentration at which httQ91-mCherry forms IBs. U2-Q91 were transiently transfected with GFP or wtFluc-GFP for 24 h. After addition of 1 µg/ml dox for 72 h, cells expressing low or high levels of wtFluc-GFP or GFP were analyzed by PulSA. Microscopy in A is representative of n > 10, and B is representative of n > 20 independent observations. Flow cytometry in C–E are representative experiments of at least two independent repeats.