Ub recruitment and UPS substrate stabilization are delayed after IB formation. (A) U2OS cells stably expressing YFP-Ub were transiently transfected with N-htt(Q91)–chFP and followed by time-lapse two-color fluorescence video microscopy (Video 2). Sequential frames show the formation of the N-htt IB (left hand column) and the complete recruitment of chFP fluorescence by 33 min. Note that during the initial formation of the IB (6- and 9-min time points), YFP fluorescence is excluded from the IB and is recruited there only after a ∼40-min delay. The arrowheads indicate the site of the forming IB. (B) U2OS cells stably expressing Ub^G76V-GFP were transiently transfected with N-htt(Q91)–chFP and monitored by time-lapse two-color fluorescence video microscopy at 20-min intervals. Selected frames of the recorded image series (Video 3) are presented. The outlined cell formed an IB at t = 0, and Ub^G76V-GFP became detectably stabilized in the cell after an ∼17-h delay. Bars, 25 µm.