Proteasome Activity Assay Protocol

In vitro assay of 26S proteasome activities was performed as previously described^{10 (link)}. Cells were collected in proteasome activity assay buffer (50 mM Tris-HCl (pH 7.5), 250 mM sucrose, 5 mM MgCl₂, 0.5 mM EDTA, 2 mM ATP and 1 mM dithiothreitol) and lysed by passing 10 times through a 27 gauge needle attached to a 1 ml syringe. Lysate was centrifuged at 10,000g for 10 min at 4°C. 15–25 μg of total protein of cell lysates were transferred to a 96-well microtiter plate (BD Falcon) and then the fluorogenic substrate was added to lysates. For measuring the chymotrypsin-like activity of the proteasome we used either Z-Gly-Gly-Leu-AMC (Enzo) or Suc-Leu-Leu-Val-Tyr-AMC (Enzo). We used Z-Leu-Leu-Glu-AMC (Enzo) to measure the caspase-like activity of the proteasome and Ac-Arg-Leu-Arg-AMC for the proteasome trypsin-like activity. Fluorescence (380-nm excitation, 460-nm emission) was monitored on a microplate fluorometer (Infinite M1000, Tecan) every 5 min for 1 h at 37°C. Protein concentration of the cell homogenates was determined using the BCA protein assay (Pierce).