Fig. 4
The FAT10 UBDs are loosely folded and degraded less efficiently when replaced by Ub. a Melting temperatures are plotted in a bar diagram for WT ΔN FAT10, the C0 ΔN FAT10 mutant, and ubiquitin and were obtained from the respective maxima of the first derivative curves of three independent differential scanning fluorimetry measurements where the temperature was varied in 0.036 °C steps from 20 to 95 °C. The data represent mean ± standard deviations (s.d.). The individual data points are shown in Supplementary Fig. 7a. b Schematic presentation of WT FLAG-FAT10 and the two FAT10-ubiquitin hybrids (FLAG-FAT10-Ub and FLAG-Ub-FAT10), where either the N- or the C-domain of FAT10 was exchanged with lysine-less Ub (K0) to prevent Ub chain formation. To suppress cleavage of Ub(K0) from the FAT10 C-domain in Ub-FAT10, Ub(K0) was expressed without the di-glycine motif. c Western blots showing bulk conjugates of WT FAT10, Ub-FAT10 or FAT10-Ub proteins, and USE1 auto-FAT10ylation. d Degradation of monomeric WT FAT10, Ub-FAT10, and FAT10-Ub was monitored by transient transfection of HEK293 cells with the indicated expression constructs. 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. c, d HEK293 cells were harvested and lysed 24 h after transfection. Cleared protein lysates were used for immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. The upper panels show the bulk conjugates in the immunoprecipitation, lower panels show the expression of the proteins in the lysates. β-actin was used as loading control. One representative experiment out of three experiments with similar outcomes is shown. e ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated FLAG-FAT10 and FLAG-tagged FAT10-Ub chimeras in the cells. The 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. Shown are the values of three independent experiments with similar outcomes as means ± s.e.m.
