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. Author manuscript; available in PMC: 2009 Sep 16.
Published in final edited form as: Science. 2009 Jun 5;324(5932):1327–1330. doi: 10.1126/science.1172871

Fig. 3.

Fig. 3

Rhes enhances sumoylation. (A) Rhes interacts with Ubc9. HEK293 cells were transfected with GST or GST-Rhes and Myc-Ubc9. After 48 hours, cells were lysed, precipitated with GSH beads and probed for Myc. Purified GST or GST-Rhes was incubated with purified Ubc9 and precipitated with GSH beads. The precipitates and inputs were immunoblotted for Ubc9. (B) Rhes sumoylates mHtt. Four μl of in vitro translated mHtt (control) and its K6,9,15,91R mutant were subjected to sumoylation [1× RB buffer, 250 ng E1, 125 ng E2, 1.5 μg SUMO1, 5 mM adenosine triphosphate (ATP), and 2 mM dithiothreitol (DTT)] in the presence of 500 ng Rhes (+) or bovine serum albumin (BSA) (−). Htt was detected by N-Htt antibody. Rhes sumoylates Htt in a time- (Rhes, 500 ng) and concentration-dependent manner. (C and D) Sumoylation of multiple substrates. Indicated substrates (500 ng) were subjected to sumoylation assay as in (B). Rhes sumoylates substrates in a (C) time- (Rhes, 200 ng) and (D) concentration-dependent manner.