Proteasome Activity Assay in Worms

Proteasome activity (chymotrypsin-like activity) was measured in worms using a previously described method [76 (link)]. Briefly, using a Precellys 24 homogenizer, worms were lysed in a proteasome activity assay buffer containing 50 mM Tris-HCl (pH = 7.5), 250 mM sucrose, 2 mM adenosine triphosphate (ATP), 5 mM MgCl₂, 1 mM dithiothreitol, and 0.5 mM ethylenediaminetetraacetic acid (EDTA). The lysate was centrifuged at 10,000× g for 15 min at 4 °C. For each test, 25 μg of total lysate was loaded into each well of a 96-well microtiter plate, after which fluorogenic substrate was added. Suc-Leu-Leu-Val-Tyr-AMC (Sigma-Aldrich, St. Louis, MO, USA) was used as a substrate for testing the chymotrypsin-like activity of the proteasome. After incubation for 1 h at 25 °C, fluorescence (excitation wavelength = 380 nm, emission wavelength = 460 nm) was measured with a SpectraMax M2 Microplate Reader (Molecular Devices, Silicon Valley, CA, USA).