The activity assay of NGLY1 was carried out as previously described [28]. Briefly, the reaction mixture containing 25 µl of the NGLY1 fraction in a total volume of 30 µl of 10 mM Tris–HCl (pH 7.5), 50 mM sucrose, 1 mM DTT, 1 mM Pefabloc™ SC, and 1 × cOmplete™ protease inhibitor cocktail (EDTA-free) together with 53 pmol of BODIPY-asialoglycopeptide (BODIPY-ASGP) was incubated at 25 °C for 6 h. The reaction was terminated by adding 100 µl of 100% EtOH and centrifugation at 20,000×g for 2 min. The resulting supernatant was collected and evaporated to dryness in a Speed-Vac concentrator. The substrate and reaction product were separated by HPLC using an InertSustain C18 HP (3 µm, 3.0 × 150 mm, GL Science; 5020-07425). The elution conditions were as follows: eluent A, DW containing 0.1% Trifluoroacetic Acid (TFA); eluent B, 100% acetonitrile containing 0.1% TFA. The column was equilibrated with eluent A/eluent B (60/40) at a flow rate of 0.45 ml/min. After injecting a sample, the concentration of eluent B was increased linearly from 40 to 95% over 25 min. BODIPY-ASGP was detected by measuring fluorescence (λ excitation 503 nm, λ emission 512 nm).

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