The activity of USP30 was determined by the increase in fluorescence measured as a result of the enzyme catalyzed cleavage of the fluorogenic substrate Ubiquitin-AMC (BostonBiochem, Cambridge, MA, USA) generating Ubiquitin and AMC. USP30 enzyme assay was performed by preincubating 20 nM of USP30 with the indicated concentration of aumdubin in the assay buffer (50 mM Tris-HCl [pH 7.5], 250 mM sucrose, 5 mM MgCl2, and 1 mM PMSF) for 15 min and then incubated with Ub-AMC substrate in a 100 μL reaction volume for 1 h at 25 °C. AMC released from substrate cleavage was recorded with a microplate reader (Varioskan Flash 3001, Thermo).
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