Enzyme assay of O-fucose, fringe, and O-GlcNAc transferases modifying epidermal growth factor domains ~In vitro O-GlcNAc transferase assay

 S2 cells were pelleted, washed with PBS, and resuspended in 1 mL of ice-cold PBS containing 1 mM phenylmethylsulfonyl fluoride (PMSF).

 The cell suspension was then placed in a nitrogen cavitation apparatus, and exposed to N₂ at 400 psi for 30 min.

 After release of the pressure, which disrupts the cells, the sample was centrifuged in a ST-720M rotor (Kubota) at 3,000 rpm (1,500 × g) for 5 min to remove nuclei and remaining whole cells.

 The supernatant was collected, and centrifuged in a TLS-55 (Beckman) at 40,000 rpm (100,000 × g) for 1 h at 4°C.

 The pellet was resuspended in ice-cold 50 mM HEPES-NaOH (pH 7.0), and stored at -80°C in small aliquots until used for O-GlcNAc transferase assay.

 Before the reaction, UDP-[³H]GlcNAc was dried under vacuum (Speed-Vac) and resuspended in water.

 For the assay, 1.6 μM UDP-[³H]GlcNAc (60 Ci mmol^-1), 2 μg of EGF20-V5His, and 0.2 μg of membrane fraction proteins were mixed in the glycosylation buffer.

 After incubation for 2 h at 25°C, the reaction was stopped by addition of 1 mL of ice-cold 50 mM EDTA.

 Discovery DSC-18 SPE tubes were conditioned by loading 1 mL of 80% acetonitrile, 0.052% TFA, and then equilibrated with 1 mL of H₂O.

 The sample was loaded to the tube and washed with 5 mL of H₂O, and the labeled substrates were then eluted with 2 mL of 80% acetonitrile, 0.052% TFA.

 Radioactivity in the eluate was measured using a liquid scintillation counter.