Metabolites were extracted from snap-frozen tissue. About 10 mg of tissue was weighed in and stored on dry ice. Ice-cold methanol/acetonitrile/water (800 μl; 2:2:1, by volume) was added per 10 mg of tissue. Tissue was homogenized in a methanol/acetonitrile/water mixture using a bead-beating procedure (glass beads, for 30 s), and the homogenate was transferred to a new tube. The beads were washed with 200 μl of methanol/acetonitrile/water (2:2:1, by volume), and the homogenate was incubated for 2 hours at −20°C. The insoluble pellet was spun down by centrifugation at 4°C (16,000g, 20 min) in a tabletop centrifuge. The pellet was saved for protein determination. Supernatants were dried down in a speed vacuum at 4°C. Pellets were also dried down. Last, pellets were resuspended using an amount proportional to the protein amount of insoluble pellet using 1:1 (v/v) acetonitrile/water before analysis. Metabolite extracts were directly measured using untargeted and targeted metabolomic analysis. Protein pellets were dissolved using a 2% SDS buffer containing 10 mM tris (at 95°C) and measured using a commercial bicinchoninic acid (BCA) assay (Thermo Fisher Scientific).

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