Immune-Complex-Based In Vitro Deubiquitination Assay

Deubiquitinases (DUBs) are attractive therapeutic targets within the ubiquitin-proteasome system, in part because four of the five DUB subfamilies are cysteine proteases amenable to the development of potent, selective inhibitors, as recently demonstrated for USP7. Identifying DUBs that deubiquitylate and stabilize specific human oncogenic proteins is therefore a promising approach to discovering new mechanism-based targets for cancer therapy. Several complementary experimental strategies are typically required to identify bona fide DUB-substrate pairs. Here, we present an efficient, straightforward in vitro immune-complex protocol to validate USP17-mediated deubiquitylation of the transcriptional co-activator β-catenin. In this assay, both β-catenin and USP17 are immunopurified from transiently transfected 293T cells and then combined to assess USP17 enzymatic activity. The protocol describes the in vitro enzymatic assay performed on immunopurified complexes and the immunoblot-based readout. It can be readily adapted to other DUBs and substrates for mechanistic studies. Key features • Requires prior experience with cell culture, immunoprecipitation, and immunoblot analysis. • Uses plasmids encoding epitope-tagged DUB (here, USP17), the substrate of the DUB of interest (here, β-catenin), and ubiquitin, which are transiently expressed in 293T cells. • Expression in mammalian cells preserves DUB (here, USP17) essential post-translational modifications. • Adaptable to other DUB-substrate pairs.