Western Blotting for Coronavirus Protein

Western blotting was used to measure the level of coronavirus protein in cells and media, as described previously [24 (link), 26 (link)]. Briefly, cells and media were collected and resuspended in cell lysis buffer (150 mM NaCl, 50 mM HEPES (pH 7.5), and 1% NP40) containing a protease inhibitor cocktail (Roche, Basel, Switzerland). Cell lysates were resolved by SDS-PAGE and transferred to immune-blot PVDF membrane filters (Bio-Rad, Hercules, CA, USA). Viral proteins were detected with a 1:5000 dilution of primary HCoV-OC43 antibody using an ECL Western blotting substrate (Dogen, Seoul, Korea). The images were acquired using the ChemiDoc Imaging System (Bio-Rad). The HCoV-OC43 antibody was purchased from Sigma-Aldrich.