Anti spike antibody

All primary antibodies were purchased from Cell Signaling Technologies except the anti-Spike antibody (Novus Biologicals) and the anti-nucleocapsid, anti-tubulin, and anti-GAPDH (Thermo Fisher) antibodies. Horseradish peroxidase (HRP)-conjugated secondary antibodies were purchased from Jackson ImmunoResearch. Protein pellets were lysed in an NP-40 lysis buffer as described earlier (33 (link)). Protein quantification was done using the bicinchoninic acid (BCA) method (G Biosciences). The immunoblots were developed on a Bio-Rad Chemidoc MP system using ECL reagents (Thermo Fisher and G Biosciences). Quantification was performed using ImageJ.

The cells were stained as previously described [29 (link),30 (link)]. In short, Vero-E6 cells were seeded in coverslips and after 48 and 72 h of infection, were fixated using 3.7% formaldehyde. Cells were rinsed three times with PBS containing 0.1 mM CaCl₂ and 1 mM MgCl₂ (PBS/CM) and then permeabilized with 0.1% Triton X-100 plus 0.2% BSA in PBS/CM for 10 min (PBS/CM/TBSA). Cells were stained with rabbit polyclonal antibody anti-spike antibody (#56578—NOVUSBIO) at 1:250 dilution for overnight, followed by a rabbit anti-IgG Dylight 550 at 1:1000 dilution for 1 h. The coverslips were mounted in slides using an antifade mounting medium (VECTASHIELD^®, Burlingame, CA, USA). Nuclear recognition was based on DAPI staining (1 μg/mL) for 5 min. Fluorescence was analyzed by fluorescence microscopy with an 100× objective lens (Olympus, Tokyo, Japan).