Proteasome Activity Assay in Worms

We used the previously described method to determine the proteasome activity (chymotrypsinlike activity) in worms [31 (link)]. Worms were first lysed by using a Precellys 24 homogenizer and proteasome activity assay buffer [50 mM Tris-HCl (pH = 7.5), 250 mM sucrose, 2 mM ATP, 5 mM MgCl₂, 1 mM dithiothreitol, and 0.5 mM EDTA]. The lysate was then centrifuged at 10,000× g at 4 °C for 15 min. For each sample, 25 μg of total lysate was added to each well of a 96-well microtiter plate, and then the fluorescent substrate Suc-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich, St. Louis, MO, USA) was added. After the plates were incubated for 1 h at 25 °C, the fluorescence was measured with a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley, CA, USA) (λex = 380; λem = 460 nm).