Figure 5. hHR23A has no effect over the ubiquitin hydrolase activity of ataxin-3.

Recombinant human (Q22) wild-type (A, C) or catalytic inactive (C14A) (E) ataxin-3 (100 nM) was incubated alone or together with recombinant hHR23A (100 nM) in a buffered solution containing hexa-ubiquitin chains (250 nM) with K63 (A, E) or K48 (C) linkages for 20 hours at 37°C. Samples were collected at 0, 2, 5 and 20 hours and analyzed by immunoblotting. The reaction products were detected using an antibody against ubiquitin (A, C, E). The appearance of reaction products resulting from the cleavage of K63-linked (B) or K48-linked (D) hexa-ubiquitin chains by ataxin-3 in the absence (light grey squares) or presence (dark grey triangles) of hHR23A was quantified; the mean ± SEM of 4 independent experiments was plotted in graphs.