Stabilization of proteasome reporters by aggregation-prone N-htt is not caused by direct competition for 26S. (A) Short-lived proteasome substrates, but not N-htt, compete for degradation of chFP-CL1 degradation. HEK293 cells stably expressing chFP-CL1 were transfected with GFP-CL1, cODC-GFP, GFP, N-htt(Q25)–GFP, or N-htt(Q91)–GFP and analyzed by two-color flow cytometry. (inset) Double logarithmic plot shows that cODC-GFP and GFP-CL1 data fit well with a linear least-squares regression (r² > 0.98), whereas N-htt(Q91)–GFP does not. The data shown are from a single representative experiment out of two independent repeats. (B) Concentration of Ub, N-htt, and Ub-associated N-htt in HEK293 cells sorted on the basis of N-htt(Q91)–chFP fluorescence intensity. HEK293 cells stably expressing Ub^G76V-GFP and transfected with N-htt(Q91)–chFP were sorted based on high and low N-htt(Q91)–chFP fluorescence intensity followed by Ub AQUA-MS analysis to determine the amount of each species in the two sorted populations. All data are represented as means ± SEM (n = 3).