Reduced glutathione

For purification of HFGF-CD59, approximately 1 × 10⁸ HEK293, HEK293-B3GALT4 KO and HEK293-B3GALT4-PIGZ DKO cells stably expressing HFGF-CD59 were treated with 0.5 unit/mL PI-PLC (Thermo Scientific) in 5 mL of PI-PLC buffer (Opti-MEM containing 10 mM HEPES-NaOH (pH 7.4), 1 mM EDTA and 0.1% BSA (Nacalai Tesque) at 37 °C for 2.5 h. HFGF-CD59 was also prepared from vector- or 3HA-B3GALT4-transfected HEK293-B3GALT4-PIGZ DKO cells. Supernatants were loaded on the columns filled with 0.25 mL of Glutathione Sepharose 4B (GE healthcare) at 4 °C. After washing with 5 mL PBS, HFGF-CD59 was eluted by 5 mL elution-buffer A (PBS containing 30 mM HEPES-NaOH (pH 7.4) and 20 mM reduced glutathione (Wako)). 100% cold trichloroacetic acid (Wako) was added to eluted samples to final 10 % followed by incubation on ice for 30 min. Proteins were precipitated by centrifugation at 12,000 × g for 15 min at 4 °C. After twice washing with 100 % cold ethanol, pellets were resolved in 1 × SDS-sample buffer with 5% β-mercaptoethanol and boiled at 60 °C for 1 h. Samples were subjected to SDS-PAGE, Coomassie brilliant blue staining (Imperial Protein Stain, Thermo Scientific), and in-gel digestion with trypsin.

Indoxyl sulfate was purchased from Biosynth (Staad, Switzerland). Phenyl sulfate and p-cresyl sulfate were synthesized at Eiweiss (Shizuoka, Japan). Indoleacetic acid was purchased from Tokyo chemical industry (Tokyo, Japan). Hydrogen peroxide and reduced glutathione were purchased from Wako Pure Chemical Industries (Osaka, Japan). Buthionine sulfoximine was purchased from Cayman Chemical (Ann Arbor, MI, USA). Meta-phosphoric acid was purchased from Sigma-Aldrich (St. Louis, MO, USA).