FIGURE 6:

Catalytically inactive YOD1 traps polyubiquitinated proteins. (A) For purification of YOD1, cells transfected with either FLAG-WT YOD1 or FLAG-C160S YOD1 were lysed in a buffer containing 1% NP-40 with or without 1% SDS. The resulting WCLs were diluted 10-fold and incubated with FLAG antibody–conjugated beads; the precipitated material was eluted with a FLAG peptide and subjected to SDS–PAGE followed by Coomassie staining or immunoblotting with the indicated antibodies. (B) Transfected FLAG-WT YOD1 or FLAG-C160S YOD1 were precipitated, and the samples were immunoblotted with the indicated antibodies. Quantification of the Hrd1 band intensity is as in Figure 2E. Data represent the mean of three independent experiments. Error bars: ±SD.