Skip to main content
. 2012 Sep 6;7(9):e43563. doi: 10.1371/journal.pone.0043563

Figure 2. VCP/p97 enhances deubiquitinase activity of ataxin-3 in vitro.

Figure 2

K63-linked (A) and K48-linked (C) hexa-ubiquitin chains (250 nM) were incubated with recombinant human (Q22) ataxin-3 (100 nM) with or without recombinant human VCP/p97 (100 nM) for 20 hours at 37°C. Samples collected at 0, 2, 5 and 20 hours were submitted to SDS-PAGE analysis and reaction products were detected by immunoblotting with an anti-ubiquitin antibody. The graphs summarize the mean ± SEM of the increase in reaction products over time resulting from the hydrolysis of K63-linked (B) and K48-linked (D) hexa-ubiquitin chains by ataxin-3 in the absence (light grey squares) or presence (dark grey triangles) of VCP/p97 in 3–5 independent experiments. Statistical analysis: * p<0.05, compared to recombinant human wild-type ataxin-3 alone at 20 hours. (E) K63-linked hexa-ubiquitin chains (250 nM) were incubated with recombinant wild-type or catalytically inactive (C14A) ataxin-3 (100 nM) in the presence of VCP/p97 (100 nM) for 20 hours at 37°C. The 0, 2, 5 and 20 hours time-points were analysed by western blotting with an anti-ubiquitin antibody.