Suc llvy amc

Cerebella or MEFs were lysed in UPS buffer (10 mM Tris, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 1.5 mM MgCl₂, 0.05% NP40, 5 mM DTT, and 2 mM ATP). Proteasome activity was determined by incubating equal amounts of protein with 1 μM fluorescent proteasome substrates N-succinyl-Leu-Leu-Val-Tyr-7-amino-4-methylcoumarin (Suc-LLVY-AMC: Sigma), Boc-Leu-Ser-Thy-Arg-AMC (Boc-LSTR-7-AMC: Sigma), or Z-Leu-Leu-Glu-AMC (Z-LLE-AMC: Sigma) as substrates for chymotrypsin, trypsin, and caspase-like activities of the proteasome, respectively, with or without 1 nM MG132 (Sigma) for 30 min at 37°C. Data reflect kinetics of the linear phases of the curves of florigenic substrate production measured on a Perkin Elmer Victor2 multiwell plate reader.

After experimental myotube incubation, cells were lysed on ice with a homogenization buffer (50 mmol Tris-HCl/L, pH = 7.5, 1 mmol EDTA/L, 5 mmol MgCl₂/L, 0.1 mmol dithiothreitol/L, 10% glycerol). After centrifugation and before measurement of protease activities, protein content was measured by spectrophotometry (Nanophotometer NP 80, Implen^®, Germany). Chymotrypsin-like activity of the proteasome fraction was measured using the fluorogenic substrate SUC-LLVY-AMC [succinyl-Leu-Val-Tyr-7-amido-4-methylcoumarin; AMC)] (Sigma-Aldrich^®) [37 (link)]. Then, 10 µL of the supernatant fluid (∼10 μg protein) was incubated in 100 μL of buffer (50 mmol Tris-HCl/L, pH 7.5, 1 mmol ATP/L, 5 mmol MgCl₂/L, and 150 μmol LLVY/L) in microplates. Standard curves were prepared using the AMC. Fluorescence was measured continuously over 1 h at 37 °C on a SpectraMax^® i3X system (Molecular devices^®) at λex = 380 nm and λem = 460 nm. Proteolytic activity was calculated from the increment of the curves from samples and standards and were expressed as pmol of AMC released/μg protein per minute.

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).

Fangchinoline with a purity more than 98% and tetrandrine with a purity more than 98% were both purchased from Shanghai Source Leaf Technology Co., Ltd. (Shanghai, China). Fangchinoline or tetrandrine was dissolved in DMSO to the concentration of 0.1 M as stock solution and stored at -20°C. Fluorogenic peptide substrates Z-LLE-AMC for checking proteasome caspase-like (C-L) activity and Z-ARR-AMC for checking proteasome trypsin-like (T-L) activity were from Calbiochem, Merck. Fluorogenic peptide substrate Suc-LLVY-AMC for checking proteasome chymotrypsin-like (CT-L) activity was from Sigma-Aldrich. Other chemical reagents, except where specially noted, were purchased from Sigma-Aldrich.