Fig. 3
The presence of the FAT10 linker is crucial for FAT10 conjugation. a Scheme showing the amino acid substitutions or deletion of the WT FAT10 linker sequence (KPSDE). The different linker mutants 5x Ala, 5x Pro, 5x Gly, and Δ-linker were generated by site-directed mutagenesis of the expression plasmid pcDNA3.1-His-3xFLAG-FAT10. The His-3xFLAG tag is referred to as FLAG in the text as well as in the figures. b HEK293 cells were transiently transfected with expression constructs for the indicated proteins to monitor proteome-wide and USE1-specific conjugation of WT as well as of mutant FAT10. Uncropped blots are shown in Supplementary Figure 8. c The degradation rate of monomeric FAT10 in the lysate as well as of FAT10 conjugates was monitored in HEK293 cells expressing the different FLAG-tagged FAT10 linker variants. Cells were treated for 2.5 or 5 h with 50 µg/mL CHX to inhibit de novo protein synthesis. Where indicated, cells were additionally treated with 10 µM proteasome inhibitor MG132 for 6 h. b, c HEK293 cells were harvested and lysed 24 h after transfection. Cleared protein lysates were subjected to immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. Proteins were separated on 12.5% Laemmli gels and visualized by western blot analysis with a monoclonal FLAG-reactive antibody (clone M2) or a USE1-reactive polyclonal antibody, as indicated. β-actin was used as loading control. One experiment out of three experiments with similar outcomes is shown. d Quantification of the amount of monomeric and of conjugated FLAG-FAT10 linker variants in c. The enhanced chemiluminescence (ECL) signals were quantified with Image Lab 4.1. software (BioRad) and normalized to signals of the loading control β-actin. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Values are shown for three independent experiments with similar outcomes as means ± s.e.m.
