Western blot analysis using C. elegans samples was performed with synchronized L4 or young adult animals, which were harvested, washed three times using M9 buffer, and centrifuged at 1400 rpm for 30 s. The worms were shock-frozen in liquid nitrogen, mixed with SDS sample buffer, and boiled for 10 min. The samples were subsequently vortexed for 10 min and centrifuged for 30 min at 15,700g at 4°C, and the supernatant was collected. The worm protein samples were electrophoresed using 8% SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. The membrane was blocked with TBS-T [20 mM Tris-HCl (pH 7.6), 140 mM NaCl, and 0.1% Tween 20] buffer containing 5% bovine serum albumin for 1 hour at room temperature and incubated with primary antibodies against p-AMPKα (1:2000; 2531S, Cell Signaling Technology, MA, USA) or α-tubulin (1:1000; sc-32293, Santa Cruz Biotechnology, TX, USA) overnight at 4°C. Anti-rabbit (1:15,000; Thermo Fisher Scientific, MA, USA) or anti-mouse (1:5000; Thermo Fisher Scientific, MA, USA) secondary antibodies conjugated with horseradish peroxidase were used to detect anti-AMPKα or anti–α-tubulin primary antibodies, respectively. The membrane was incubated in the chemiluminescent horseradish peroxidase substrate (Thermo Fisher Scientific, MA, USA), and the signal was detected by using x-ray film (Agfa, Antwerp, Belgium). Western blot analysis using cultured human cells was performed as previously described (38). Western blot data were quantified using ImageJ software.

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