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. Author manuscript; available in PMC: 2013 Aug 1.
Published in final edited form as: Nat Chem Biol. 2012 Dec 9;9(2):112–118. doi: 10.1038/nchembio.1140

Figure 1. Hip increases client protein ubiquitination and promotes AR112Q clearance.

Figure 1

(a) Hip promotes nNOS ubiquitination. HEK293T cells transiently expressing nNOS, HA-ubiquitin (HA-Ub) and increasing amounts of Hip were treated with lactacystin (24 hr). nNOS was immunoprecipitated from cytosolic lysates, and Ub-nNOS from three separate experiments was quantified (mean ± SEM). ***P<0.001. (b, c) Hip decreases AR112Q levels. (b) HeLa cells transiently expressing AR112Q and increasing amounts of Hip were treated with R1881 (10 nM) for 24 hr. Lysates were separated into supernatant and 15,000 g pellet fractions, then analyzed by western blot (left) (For uncropped gel images, see Supplementary Figure 12). At right, pelleted AR from three experiments was quantified. n.s. = not significant.(c) HeLa cells were cotransfected with AR112Q and Hip, then treated with R1881 (10 nM) for 24 hrs prior to 8 hr treatment with MG132 (10 μM). (d) Hip diminishes AR112Q aggregates. PC12 cells expressing tet- regulated AR112Q were transfected with FLAG-Hip, then treated with doxycycline and R1881 (10 nM) for 24 hr. AR and FLAG-Hip were visualized by confocal microscopy (left). Scale bar, 10 μm. The expression of Hip significantly decreased the frequency of polyQ AR intranuclear inclusions (right, mean ± SEM).