26S Proteasome Activity Assay

The proteasome assay was performed according to previously reported protocols⁴². In the absence of proteasome inhibitors, cell lysates were prepared through ultra-sonication in retic buffer (30 mM Tris (pH 7.8), 5 mM MgCl_2, 5 mM KCl, 0.5 mM DTT, 2 mM ATP). In 96-well black/clear plates (BD Falcon, USA), 5 μg of the cytoplasmic proteins were treated with 200 μM of the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (SUC-LLVY-AMC; Enzo, Republic of Korea) to a final concentration of 100 μM. Cytoplasmic proteins were treated with the fluorogenic substrate and incubated for up to 4 hrs at 37 °C. After the incubation period, the reaction was stopped with the addition of cold ethanol, and the fluorescence of the samples was measured at 380/460 nm (Ex/Em) using a fluorometer (Glomax, Promega, USA). The following fluorogenic substrates were obtained from Enzo and were also used in this study: Bz-Val-Gly-Arg-AMC (Bz-VGR-AMC; trypsin-like activity), Ac-Gly-Pro-Leu-Asp-AMC (Ac-GPLD-AMC; caspase-like activity), and ubiquitin-AMC (deubiquitinating activity).