The spike glycoprotein of SARS-CoV-2 in cells and on pseudovirions were detected by using western blot. Briefly, cells transfected with plasmids encoding spike glycoprotein of SARS-CoV-2, SARS-CoV, MERS-CoV, and MHV were lysed at 40 h post transfection by RIPA buffer (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% NP40, 1×protease inhibitor cocktail). To pellet down pseudovirions, the viral supernatants were centrifuged at 25,000 rpm for 2 h in a Beckman SW41 rotor at 4 °C through a 20% sucrose cushion, and virus pellets were resuspended into 30 μl RIPA buffer. The samples were boiled for 10 min and separated in a 10% SDS-PAGE gel and transferred to nitrocellulose filter membranes. After blocked by 5% milk, the membranes were blotted with primary antibodies, followed by incubated with horseradish peroxidase (HRP) conjugated secondary antibodies (1:5000) and visualized with Chemiluminescent Reagent (Bio-Rad). MHV S proteins were detected using polyclonal goat anti-MHV S antibody AO4 (1:2000); SARS-CoV S proteins were blotted with either polyclonal anti-SARS S1 antibodies T62 (1:2000) (Sinobiological Inc, Beijing, China) or mouse monoclonal against SARS S1 antibody MM02 (1:1000) (Sinobiological Inc, Beijing, China), MERS-CoV and SARS-CoV-2 S proteins were detected using mouse monoclonal anti-MERS S (1:1000) (Sinobiological Inc, Beijing, China) and anti-FLAG M2 antibody (1:1000) (Sigma, St. Louis, MO, USA), respectively.

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varvara ivanovna Dyatlova
2022-04-30 06:20:07 Reply

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