Assessing Proteasome Activity in Nematodes

Worms were synchronized and cultured on NGM agar plates containing 1% ethanol or without ethanol at 16°C for 48 h. After increasing the incubation temperature to 25°C for 40 h, worms were obtained and washed three times with M9 buffer. Then, worms were frozen at −80°C and lysed by sonication with lysis buffer (50 mM HEPES pH 7.5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100). The concentration of extracted protein was determined using Bioepitope Bicinchoninic Acid Protein Assay Kit (Bioworld, United States). Proteasome chymotrypsin-like activity was assayed using the fluorogenic peptide Suc-LLVY-AMC (Sigma-Aldrich, Germany). The soluble protein was reacted with proteasomal activity assay buffer containing 50 mM HEPES pH 7. 5, 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, 2 mM ATP, 2 mM DTT, and 25 μM Suc-LLVY-AMC at 37°C for 30 min in the dark. The fluorescence level was measured at an excitation wavelength of 340 nm and an emission wavelength of 465 nm using a microplate reader (Infinite 200 PRO, TECAN, Switzerland). The proteasome activity was calculated as the difference between total activity and residual activity with 5 μM MG132 (Aladdin, Shanghai, China), which was used to inhibit proteasome activity (Dammer et al., 2011 (link); Vilchez et al., 2012 (link); Shashova et al., 2014 (link)). The experiment was performed thrice (approximately 1,000 nematodes per group).