Preparation of cytosolic fraction and enrichment of NGLY1

For preparation of lysate, 50 mg of rat brain was sliced and resuspended in 500 µl NGLY1 buffer (5 mM Tris–HCl; pH 7.5), 250 mM glycerol, 1 mM Pefabloc™SC (Sigma-Aldrich; 11429868001), and 1 × cOmplete™ protease inhibitor cocktail (Sigma-Aldrich; 11836145001). Tissue was homogenized using a Biomasher-II (Nippi-inc; 320 103) by 4 × 20 s homogenization with intervals of 20 s on ice. The sample was centrifuged at 20,000×g for 5 min at 4 °C and the supernatant was transferred to a new tube. The sample was further separated by ultracentrifugation (100,000×g for 30 min at 4 °C) and the supernatant was collected as a cytosolic fraction. Enrichment of NGLY1 from the cytosolic fraction was performed using a Butyl-Sepharose column (GE Healthcare Life Sciences; 17-0980-01) [45]. Briefly, saturated ammonium sulfate solution was added to the cytosolic fraction until the ammonium sulfate concentration reached 220 mM. The suspension was then centrifuged at 20,000×g for 10 min at 4 °C and the supernatant was collected. Next, the supernatant was applied to a Butyl-Sepharose column conditioned with equilibration buffer (NGLY1 buffer containing 220 mM ammonium sulfate). The column was washed with equilibration buffer and washing buffer (NGLY1 buffer containing 175 mM ammonium sulfate). Finally, NGLY1 was eluted with 5 ml NGLY1 buffer. The eluted fraction was concentrated using an Amicon Ultra-15 (30,000 molecular weight cut-off; Millipore; UFC903008). The protein concentration was measured using a BCA assay kit (Thermo Scientific; 23227) according to the manufacturer's instructions.