Immune blot pvdf membrane filters

For the Western blot analysis, cells were collected in cell lysis buffer (150 mM NaCl, 50 mM HEPES [pH 7.5], and 1% NP40) containing protease inhibitor cocktail (Roche, Basel, Switzerland). Polypeptides in whole-cell lysates were resolved by SDS-PAGE and transferred onto immune-blot PVDF membrane filters (Bio-Rad, Hercules, CA, USA). Proteins were detected with a 1:1000 or 1:5000 dilution of primary antibodies using an ECL system (Dogen, Seoul, South Korea). Images were acquired using ChemiDoc-it 410 Imaging System (Analytik Jena, Upland, CA, USA) and ImageQuant LAS 4000 System (GE Healthcare, Waukesha, WI, USA). LNX1-specific antibodies were purchased from Lifespan Biosciences (Seattle, WA, USA). Cyclin D1- and cyclin E1-specific antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA), and PARP-specific antibodies were purchased from GeneTex (San Antonio, TX, USA).

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.