Figure 7.

The noncovalent interaction of FAT10 with OTUB1 stimulates its DUB activity in cellulo and in vitro. A and B, HEK293 cells were transiently transfected with HA-ubiquitin, Flag-OTUB1 and Flag-FAT10 WT, or Flag-FAT10 AV. Cell lysates were subjected to anti-HA immunoprecipitation (IP) and Western blot (IB) analysis using anti-HA and anti-Flag peroxidase–conjugated antibodies. C, lysates of HEK293 cells transiently expressing HA-ubiquitin WT, HA-ubiquitin K48only, HA-ubiquitin K63only (remaining Lys residues were mutated to Arg), Flag-OTUB1, and Flag-FAT10 were subjected to immunoprecipitation using anti-HA–coupled agarose and Western blot analysis. β-Actin was used as the loading control in A–C. D, quantitative analysis of AMC fluorescence (ex. 360 nm, em. 465 nm) of ubiquitin-AMC (5 μm) incubated with OTUB1 (10 μm) in the absence or presence of FAT10, UbcH5B, and USE1 (5 μm) for the indicated time periods at 30 °C. One representative experiment of three experiments with similar outcomes is shown.