Fig. 5

Biochemical and kinetic analysis of the USP25 assemblies. a Cartoon representation of the different truncation constructs of USP25. Left columns indicate the names and the right columns the residue range for each construct. b Size-exclusion chromatography profiles of the different USP25 truncation constructs run under identical experimental conditions in Superdex 16 column. Below, SDS-PAGE of the fractions of the gel filtration peaks. c Deubiquitinating activity assays of the different elution fractions of the gel filtration chromatography using Ub-AMC as a substrate. Activity assays were run in triplicate. d Steady-state “Michaelis–Menten” kinetic plot of the indicated oligomer constructs of USP25, including the purified tetramer, the truncated dimer construct (ΔIL) and the truncated monomer construct (ΔLCC-IL). Error bars represent the standard error for two independent experiments