ub rho110 substrate  (R&D Systems)

Journal: Biochemical Journal

Article Title: Investigation of USP30 inhibition to enhance Parkin-mediated mitophagy: tools and approaches

doi: 10.1042/BCJ20210508

Figure Lengend Snippet: ( A ) Chemical structure of USP30Inh-1, 2 and 3. ( B ) Inhibition of the activity of recombinant human USP30 (rhUSP30) protein was tested by using 100 nM Ub-Rho110 as substrate when incubated with the indicated concentrations of USP30Inh-1, 2 and 3. ( C ) The Biotin-Ahx-Ub-PA was used with the ABP assay in order to determine in-cell target engagement of USP30 inhibitors. Representative immunoblotting for the USP30 expression when SHSY5Y cells were treated with the indicated USP30Inh-1 concentrations for 24 h before incubating cell lysates with 2.5 µM Ub-PA for 1 h at room temperature. Engagement of the probe is indicated by an ∼8 kDa shift in the molecular weight of USP30. ( D ) Quantification of the USP30 target engagement for USP30Inh-1, 2 and 3 from 2–4 independent experiments (USP30Inh-1, n = 4; USP30Inh-2, n = 3 and USP30Inh-6, n = 2). ( E ) DUB selectivity assay (DUBprofiler TM ) was conducted using 1 and 10 µM of USP30Inh-1, 2 and 3. ( F ) The effect on the mono-Ub formation was assessed via SDS–PAGE where the indicated USP30Inh-1 concentrations were incubated for 2 h at 37°C with 450 nM rhUSP30 and 2.5 µM K6-di-Ub. ( G ) Changes in the di- and mono-Ub formation were quantified from three independent experiments.

Article Snippet: Selectivity profiling of 41 DUB enzymes was performed at Ubiquigent (Dundee, U.K.) using the DUBprofiler™ platform and Ub-Rho110-glycine substrate based-assay.

Techniques: Inhibition, Activity Assay, Recombinant, Incubation, Western Blot, Expressing, Molecular Weight, SDS Page