Fig. 1

In vitro ubiquitination of UNC-45 by CHN-1 and UFD-2. a Time-course analysis of UNC-45 ubiquitination by CHN-1, UFD-2, or both E3 ligases. Reactions were incubated for 15, 30, and 60 min with ATP and analyzed by anti-UNC-45 western blot. b Quantification of the western blot lane profiles at time points 0 (without ATP) and 60 min. The two plots show the overlaid profiles upon addition of CHN-1 (black), UFD-2 (orange), and CHN-1/UFD-2 (red). c Quantification of the UNC-45(Ub₂–Ub_n) signal. The corresponding (Ub₂–Ub_n) area of the reaction without ATP was used for background subtraction and is displayed as time point 0 for every reaction mix. d Ubiquitination of UNC-45 by UFD-2 using native or methylated ubiquitin (MeUb), with the latter preventing formation of poly-ubiquitin chains. Reactions were incubated for 15, 30, and 60 min with ATP and analyzed by anti-UNC-45 western blot. e Analysis of UNC-45 ubiquitination by using increasing concentrations (0.25, 0.5, 1, and 2 μM) of UFD-2 or CHN-1. To evaluate the possibility of a composite UFD-2/CHN-1 E4 activity, different amounts of CHN-1 (0.25, 0.5, 1, and 2 μM) were added to ubiquitination reactions containing 0.5 μM UFD-2. All assays were incubated for 60 min and were analyzed by anti-UNC-45 western blot