Fig. 5
Cys-replacement stabilizes FAT10 and its conjugates in vivo. a HEK293 cells expressing WT or Cys-free FLAG-tagged FAT10 were treated for 0.5, 1, or 2.5 h with 50 µg/mL CHX to monitor degradation rates. In addition, cells were treated with 10 µM proteasome inhibitor MG132 for 3.5 h, as indicated. Cells were harvested, lysed, and subjected to immunoprecipitation using EZview Red anti-FLAG-M2 affinity gel. Proteins were separated on 4–12% NUPAGE gradient gels and subjected to western blot analysis using a directly peroxidase-labeled, monoclonal FLAG-reactive antibody (clone M2). β-actin was used as loading control. One representative experiment out of four independent experiments with similar outcomes is shown. b ECL signals were quantified and graphs show the amount of monomeric as well as of conjugated WT or Cys-free FLAG-FAT10 normalized to the amount of β-actin in the lysate. The values of the untreated cells were set to 100% and the other values were calculated accordingly. Shown are the values of four independent experiments with similar outcomes as means ± s.e.m. c Molecular dynamics (MD) simulations of the N- (left) and C-domain (right) of human FAT10. Top panels: three of the most prominent structures obtained by root mean square deviation clustering from each MD simulation in cartoon representation (WT: white, Cys-free: blue). Bottom panels: Residue-wise root mean square fluctuation (RMSF) values. Arrows in the graph show position of cysteine mutations. N- and C-terminal tails are flexible. Moreover, flexibility is decreased in the N-domain in Cys-free mutants while the overall structure of both domains is preserved
