Figure 3. YM-1 increases client protein ubiquitination and diminishes AR112Q aggregation.

(a) YM-1 promotes nNOS ubiquitination. HEK293 cells stably expressing nNOS were treated with increasing amounts of YM-1 and lactacystin for 24 hours. Short exposure of input (on left) shows unmodified nNOS, while longer exposure shows an accumulation of a higher molecular weight (MW) species representing mono-ubiquitinated nNOS ²⁵ (highlighted in rectangle). (For uncropped gel images, see Supplementary Figure 12) Quantification of mono-ubiquitinated nNOS from four separate experiments is shown in the middle, **P<0.01, ***P<0.001. Immunoprecipitation (on right) shows increased nNOS poly-ubiquitination in the presence of YM-1. (b) YM-1 decreases insoluble AR112Q. PC12 cells expressing tet-regulated AR112 were treated with R1881 (10 nM) and YM-1 for 16 hours. Pelleted (15,000 g) AR112Q was visualized by immunoblot analysis and data from three separate experiments was quantified (mean ± SEM). *P<0.05, **P<0.01(c) PC12 cells were treated as in (b). AR was visualized by fluorescence microscopy and cells with nuclear inclusions quantified. Data are mean ± SEM. **P<0.01.