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. Author manuscript; available in PMC: 2016 Oct 13.
Published in final edited form as: Nature. 2016 Apr 13;532(7599):398–401. doi: 10.1038/nature17433

Figure 1. USP14 cleaves supernumerary ubiquitin chains from substrates.

Figure 1

a, Canonical model of USP14 activity. USP14 is thought to shorten ubiquitin chains progressively from their distal tip. b–f, In vitro degradation and deconjugation assays with polyubiquitinated N-terminal fragment of cyclin B1 (Ubn-NCB1; HA tagged). Cartoons show idealized representations of the expected distribution of ubiquitin moieties throughout. b, Human proteasome (Ptsm; 4 nM) was incubated with Ubn-NCB1 (~110 nM final) generated by UbcH10. Where indicated, USP14 (80 nM) was added. c, As in b, except using polyubiquitinated NCB1 generated with lysine-less Ub. d, As in b, except using polyubiquitinated conjugates on K64-only NCB1 (1KNCB1, unless otherwise noted), generated by UbcH10 and Ube2S. e, Ubn-NCB1 (~110 nM) was incubated with hexokinase-treated ADP-proteasome (ADP-Ptsm) in the presence or absence of USP14 (80 nM). f, As in e, except using polyubiquitinated K64-only NCB1. Samples were analysed by SDS–PAGE/immunoblotting for HA. For gel source data, see Supplementary Information.