Fig 1. Development of an assay to monitor protein degradation.

(A) Design of protein degradation assay. (B) 20 nM ^K48UbⁿGST–Wbp2 was incubated with 5 nM 26S proteasome at 37 ˚C for 2 hours in the absence and presence of different inhibitors (20 μM capzimin/CZM or 10 μM carfilzomib/CFZ). Reactions were fractionated by SDS-PAGE and analyzed using a Typhoon fluorescence scanner. Boxes mark the input high MW polyubiquitinated substrate and a deubiquitinated species that accumulated in reactions inhibited by CFZ (Verma et al., 2002). (C) Measurement of proteasome activity using the fluorescence polarization assay. ^K48UbⁿGST–Wbp2 (2.5 nM) was incubated with 26S proteasome (1 nM) at 37 °C in the absence or presence of different inhibitors (10 µM CZM or 1 µM bortezomib/BTZ). (D) Measurement of proteasome protein breakdown activity in cell lysate in response to increasing concentrations of carfilzomib. Shown are the reaction kinetics (upper panel) and dose-response (red curve in the bottom panel) of proteasome activity measured at 37 °C using ^K48UbⁿGST–Wbp2 as substrate and lysate from cells treated with different concentrations of carfilzomib. For comparison, a dose-response curve measured using Suc-LLVY-amc is plotted in black (bottom panel). Error bars represent s.d., n = 3 wells, from 1 representative of 3 independent experiments. See also Figure S1 and Table S1.