Western Blot Analysis of Glycoproteins

The samples were heated in 2× Laemmli buffer with 2% beta-mercaptoethanol (BME) at 100°C for 20 min and run on polyacrylamide gels (5% to 20% gradient; Bio-Rad). Gels were stained with SimplyBlue SafeStain (Invitrogen) for an hour and then destained in water for another hour. For Western blot assays, SDS-PAGE was performed with the sGPCe side by side with an irrelevant control protein (recombinant influenza A virus H7 protein). The gels were then transferred onto nitrocellulose membranes according to a previously described protocol (39 (link)). Each monoclonal antibody was used for primary staining at 30 µg/ml, and the secondary staining was performed using anti-mouse IgG (whole molecule)–alkaline phosphatase (AP) antibody produced in goat (Sigma-Aldrich) at a dilution of 1:1,000. The membrane was developed using an AP conjugate substrate kit, catalog no. 1706432 (Bio-Rad). To ensure that the proteins were transferred onto the nitrocellulose membrane, an anti-His monoclonal antibody (TaKaRa Bio Company) was used as a positive control, detecting a hexahistidine tag on the GPCs and the control protein.