Fig. 3: Assays of a panel of recombinant DUBs for Fubi-PA reactivity confirmed USP16 and USP36 as dual ubiquitin/Fubi-specific enzymes.
From: Molecular basis for ubiquitin/Fubi cross-reactivity in USP16 and USP36
a, Semisynthesis of the Fubi-PA probe from Fubi-MesNa (top) and characterization of these species by intact protein MS (bottom). b, Reactivity assessment of recombinant USP16 incubated with the indicated probes for 1 h at 37 °C. Chloroacetamide (CAA) pretreatment was performed where indicated. Protein samples were analyzed by SDS–PAGE and Coomassie staining; ABP, activity-based probe. c, Reactivity assessment of recombinant USP36 as described in b. d, Results of a DUB panel inhibition assay shown as a heat map. Recombinant DUBs were incubated with Fubi-PA (4 µM) for 15 min at room temperature, and their activities were subsequently assessed. Residual activities for USP16 and USP36 are given as numbers. Data show results from technical duplicates normalized for each DUB to its respective activity in the absence of probe; UCHs, ubiquitin carboxy-terminal hydrolases; OTUs, ovarian tumor family of DUBs. e, Reactivity assessment of recombinant USP2 as described in b and c. f–h, Thermal stability assessment of USP36 (f), USP16 (g) and USP2 (h) complexed with Fubi or ubiquitin probes. Means and individual results of three samples are plotted; Tm, protein melting temperature.
