Extended Data Figure 1. USP14 and Ubp6 are highly specific for multi-chain ubiquitin-protein conjugates.

In vitro degradation and deconjugation assays with polyubiquitinated conjugates. a, b, In vitro deubiquitination assays with Ub_n-NCB1 or Ub_n-1KNCB1 generated by Ubc4 in parallel. Each conjugate sample (~110 nM) was incubated with 4 nM of human proteasome in the presence or absence of USP14 (80 nM). c, This experiment tests whether the failure of USP14 to deubiquitinate a single-chain conjugate formed using K64-only NCB1 (Figs. 1d and 1f) is predictive of the behavior of USP14 on other single-chain conjugates. Conjugates (~110 nM) were incubated with human proteasome (4 nM) in the presence or absence of USP14 (80 nM). d, e, In vitro degradation and deubiquitination assays with polyubiquitinated Sic1^PY or polyubiquitinated K36-only Sic1^PY (1KSic1^PY). d, Ub_n-Sic1^PY (~200 nM) was incubated with human proteasome (5 nM) in the presence or absence of USP14 (100 nM). e, Assays were performed similarly as in d, except using a single-lysine variant of Sic1 (1KSic1^PY) modified with wild-type Ub. IU1 (75 μM)³ was used to inhibit USP14 deubiquitinating activity. f, In vitro degradation and deconjugation assays with polyubiquitinated securin. Human proteasome (4 nM) was incubated with Ub_n-securin (~100 nM) generated by UbcH10 and APC/C. Where indicated, USP14 (80 nM) was added. Samples were resolved by SDS–PAGE and immunoblotted using an antibody to the HA (a–c) or T7 (d, e) epitopes, and securin (f).