Phosphatase inhibitor cocktail set 3

Yeast wild-type and ERMES mutant cultures were cultured in YPGly (3% [wt/vol] glycerol, 1% [wt/vol] Bacto yeast extract, and 2% [wt/vol] Bacto peptone) at 30°C. Cells were harvested at OD₆₀₀ < 4.0, and mitochondria were purified with discontinuous Nycodenz as described (Glick & Pon, 1995 (link)). Protease inhibitor mixture (Roche Complete EDTA-free), phosphatase inhibitor cocktail set II (EMD Millipore), and phosphatase inhibitor cocktail set 3 (Sigma-Aldrich) were added to the solutions. Gradient-purified mitochondria were frozen in liquid nitrogen and stored at −80°C until further analysis. Mitochondria from yeast Δcoq mutants were purified in the same manner from cultures expanded in YPGal medium (2% [wt/vol] galactose, 0.1% [wt/vol] dextrose, 1% [wt/vol] Bacto yeast extract, and 2% [wt/vol] Bacto peptone).

Frozen brains were placed in acrylic mouse brain matrices, and 2‐mm‐thick slices were cut out using razor blades from the brain matrix. Using the Paxinos and Watson's mouse brain atlas (Paxinos & Franklin, 2012 ), the hippocampus was dissected out bilaterally. Frozen hippocampus brain samples (17 mg per sample) were homogenized in 1 ml of cold radioimmunoprecipitation assay buffer lysis buffer (RIPA); 50‐mM Tris–HCl pH 7.4, 150‐mM NaCl, 0.5% NaDOC, 1‐mM EDTA, 1% Triton, 0.1% SDS, 1‐mM Na3VO4, 1‐mM NaF, supplemented with a protease (cOmplete™ Protease Inhibitor Cocktail, Roche, cat. Number: 11836145001) and a phosphatase (Phosphatase Inhibitor Cocktail Set III, Millipore, cat. Number: 524527) inhibitor cocktail. The suspension was incubated for 2 h at 4°C, followed by centrifugation at 12,000 rpm for 15 min at 4°C. The supernatant was transferred to a new clean centrifuge tube, and the Bradford colorimetric method was used to determine the concentration of the total protein. The protein extracts were diluted 1:1 in loading buffer (Ditiotreitol [DTT] 2X) and heated for 5 min at 99°C before being subjected to electrophoresis. Natural product studies are reported in compliance with the recommendations made by the British Journal of Pharmacology (Izzo et al., 2020 (link)).