Extended Data Figure 2. Preparation of ATP-depleted proteasome for uncoupling degradation from deubiquitination.
a, For ATP-depletion, purified human proteasomes were treated with hexokinase or mock-treated with vehicle for hexokinase (i.e. water). Native gel analysis of untreated proteasome (ATP-Ptsm) or hexokinase-treated ADP-proteasome (ADP-Ptsm) with suc-LLVY-AMC and Coomassie Brilliant Blue (CBB) staining. Each lane was loaded with 5 μg of protein. Note that there was essentially no change in proteasome integrity upon hexokinase treatment. b, In vitro Ubn-NCB1 degradation assays with ATP-Ptsm or ADP-Ptsm (4 nM each). Assays were performed and analysed as in Fig. 1b or 1e.