Extended Data Figure 8. USP14 cleaves ubiquitin chains at the proximal site.

In vitro deubiquitination assays with multi-chain conjugates. a, Multi-chain K63Ub₄ conjugates on NCB1 were prepared using pre-formed K63Ub₄ free chains as described in Methods. Assays were performed as in Fig. 2a. Arrow indicates accumulated K63Ub₄-NCB1 as major deubiquitinated species generated by USP14 cleavage. Asterisks indicate presumptive deconjugated K63Ub₅- or other multi-chain K63Ub_n-NCB1 species originating from other K63Ub_n contaminants in the commercial preparation of K63Ub₄. b, Ub_n-NCB1 was generated using UbcH10, APC/C, and wild type ubiquitin, and purified as described in Methods. Note that this conjugate may be enriched with short multi-ubiquitin chains with different linkages, based on previous reports¹¹. Deubiquitination assays were performed as in Fig. 2c. Arrowhead indicates di-Ub chains as major products of USP14 activity, suggesting that Ub_n-NCB1 in this sample was rich in di-ubiquitin chains. Samples were resolved by SDS–PAGE and immunoblotted with antibody against the HA epitope (a) or Ub (b). c, TMT labelling and quantitative mass spectrometry. Purified Ub_n-NCB1 was assayed in biological duplicates for each condition, and the resulting samples were analysed for specific ubiquitin chain linkages by mass spectrometry as in Fig. 2d. The experiment was repeated in biological duplicates at a 4-min point with equivalent results (data not shown).