Fig. 1
From: The ubiquitin-like modifier FAT10 interferes with SUMO activation
JunB becomes FAT10ylated at a SUMOylation consensus site. a JunB-FAT10ylation under overexpressing conditions in HEK293 cells. Cells were transiently transfected with expression plasmids for the proteins indicated and an immunoprecipitation (IP) against HA-FAT10 combined with western blot (IB) analysis was performed. 10 μM proteasome inhibitor MG132 was added for a total of 6 h before harvesting, where indicated. β-actin was used as loading control. Shown is one experiment out of three experiments with similar outcomes. b Conjugation of FAT10 to JunB under endogenous conditions in human PBLs. Endogenous FAT10 expression was induced by IFN-γ/TNF-α treatment for 48 h and JunB-FAT10 conjugate formation was analyzed in a combined immunoprecipitation/western blot analysis. Shown is one experiment out of three experiments with similar outcomes. The asterisk marks an unspecific background band. Mouse IgG1 was used as isotype control for the IP. Bar graph represents relative expression levels of FAT10 mRNA, normalized to the housekeeping gene GAPDH as measured by real-time PCR. Ct-levels of untreated cells were 22.4, and 21.7 for IFN-γ/TNF-α treated cells. c The degradation rate of the JunB-HA-FLAG-FAT10 conjugate in transiently transfected HEK293 cells was monitored by cycloheximide (CHX) chase experiments. 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 a total of 6 h. Immunoprecipitation and western blot analysis was performed as described in a. d Densitometric analysis of ECL signals of CHX chase experiments as shown in c. ECL levels were normalized to the ECL signals of β-actin and the level of 0 h CHX was set to unity. Values are shown for five independent experiments with similar outcomes as means ± s.e.m. e FAT10 becomes conjugated to a lysine within one of the three reported SUMOylation consensus sites in JunB. Western blot analysis of JunB-HA-FLAG-FAT10 conjugates in transiently transfected HEK293 cells upon expression of wild-type JunB or of JunB mutants, in which single lysines within three different SUMOylation consensus sites or all three (JunB-K3R-HA) were mutated to arginines. Immunoprecipitation and western blot analysis was performed as described in a. Shown is one experiment out of three experiments with similar outcomes. Source data are provided as a Source Data file
