Anti sars cov 2 n

SARS-CoV-2 strain USA-WA1/2020 was obtained from BEI Resources, NIH, NIAHD (Cat # NR52281) and was plaque purified in Vero E6 cells to identify plaques lacking furin cleavage site mutations. A WT virus plaque was then propagated on Vero E6 cells stably expressing TMPRSS2 (kindly provided by Dr. Shan-Lu Liu, Ohio State University) for 72 h. The virus was aliquoted, flash-frozen in liquid nitrogen, and stored at −80C. The virus stock was titered on Vero E6 cells by TCID50 assay. For infection experiments, the virus was added to cells for 24 h. Cells were then collected by trypsinization and were either lysed with Trizol reagent for RNA extraction or were fixed with 4% paraformaldehyde in PBS for 1 h prior to staining for flow cytometry. Staining was performed with anti-SARS-CoV-2 N (Cat # 40143-MM08, Sino Biological) as described previously (28 (link), 66 (link)). Flow cytometry was performed on a FACSCanto II machine (BD Biosciences). Data were analyzed using FlowJo software.

Total cell extracts were lysed in sample buffer containing SDS, protease and phosphatase inhibitors (Roche), β-mercaptoethanol, and a universal nuclease (Fisher Scientific). Proteins were resolved on an SDS polyacrylamide gel, transferred to a polyvinylidene difluoride (PVDF) membrane, hybridized with a primary antibody, reacted with an infrared (IR) dye-conjugated secondary antibody, visualized using a Li-COR Odyssey Imager (Li-COR), and analyzed using Image Studio software. Primary antibodies used for immunoblotting included anti-SARS-CoV-2 N (SinoBiological 40143-R001) and GAPDH (Millipore-Sigma G8795) monoclonal antibodies. Secondary IR antibodies were purchased from Li-COR.