Protease inhibitor cocktail tablets

Western blotting was performed strictly according to previously described procedures [19 (link)]. Briefly, cells were washed with ice-cold PBS and soluble proteins were extracted with cell lysis buffer (100 mM Tris-HCl (pH = 8), 150 mM NaCl, 1% NP-40, phosphatase and protease inhibitor cocktail tablets (Abcam, Shanghai, China)) according to the manufacturer’s protocol. The protein concentration of each sample was determined by a BCA Kit (Beyotime, China). An equal amount of protein was separated by SDS-PAGE and transferred onto a nitrocellulose membrane (Millipore). The membrane was then blocked with 5% non-fat milk for 2 h at room temperature and incubated with primary antibodies overnight at 4 °C. Antibodies were obtained from Sigma-Aldrich (HA, # H9658) and Proteintech (Wuhan, China) (β-actin, # 60008-1-Ig). Signals were visualized using infrared imaging systems and quantified using ImageJ software v 1.0.20 (Odyssey CLX, LiCor Biosciences, Lincoln, NE, USA).

Western blot analysis was performed as previously described procedures [72 (link)]. Briefly, cells were washed with ice-cold PBS and soluble proteins were extracted with cell lysis buffer (100mM Tris-HCl pH = 8.0, 150mM NaCl, 1% NP-40, phosphatase and protease inhibitor cocktail tablets (Abcam)) according to the manufacturer’s protocol. For the analysis of S protein processing in VSV pseudotyped particles (VSVpp), we loaded 10 ml VSVpp onto 500 μl of a 20% (w/v) sucrose cushion and performed high-speed centrifugation (25000 g for 120 min at 4°C), the concentrated particles were re-suspended in 50 μl PBS. Equal amounts of protein samples were separated by 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose filter (NC) membranes. Mouse monoclonal antibodies targeting FLAG tag, β-actin (TransGen Biotech, 1:5000) were used as primary antibodies, horseradish peroxidase-conjugated (HPR) goat anti-mouse IgG antibody (Southern Biotechnology Associate, 1:10000) was the second antibody. The quantitative result of the ratio of cleaved to a full-length spike in immunoblots was analyzed by Image J software.