Rabbit anti sars cov 2 s

Immunofluorescence assays were performed to examine protein expression in virus-infected Vero cells. Vero cells were infected at MOI 0.01 with PIV5, CVXGA1, CVXGA2, CVXGA3, CVXGA5, CVXGA13, or CVXGA14 for 3 days before being fixed with 80% methanol. The cells were incubated with mouse anti-PIV5 V/P monoclonal antibody (PK 366), rabbit anti-SARS-CoV-2 S (Sino Biological catalog no. 40150-R007), or SARS-CoV-2 N (ProSci catalog no. 35–579) antibodies at 1:500 in PBS + 3% bovine serum albumin (BSA) for 1 hr. Next, the cells were washed with PBS and incubated with goat α-mouse Cy3 (KPL) or goat α-rabbit Cy3 (KPL) at 1:500 in PBS + 3% BSA for 30 mins. The cells were washed with PBS and imaged with an EVOS M5000 microscope (Thermo Fisher Scientific).

Cells were lysed in lysis buffer (10 mM iodoacetamide [Sigma-Aldrich], Complete protease inhibitor tablets [Roche], 300 mM sodium chloride, 50 mM Tris-HCl [pH 7.5], and 0.5% Triton X-100 [Sigma-Aldrich]). Virus was pelleted through 20% sucrose and resuspended in lysis buffer. Cell and virus lysates were heated to 95 °C for 5 min. Samples were analyzed on 10% 1.5 mm Tris-glycine gels, followed by transfer to PVDF membrane (BioRad) using a BioRad Trans-Blot Turbo Transfer system according to manufacturer's instructions. Blots were probed with the following antibodies: rabbit anti-SARS-CoV-2 S (Sino Biological; #40589), rabbit anti-SARS-CoV-2 S2 (Sino Biological; #40590), human anti-HIV immune globulin (HIV-Ig; NIH AIDS Reagent Program), and mouse anti-tubulin (Invitrogen; #62204). Secondary antibodies conjugated with horseradish peroxidase were used for chemiluminescent detection for SARS-CoV-2 S and other proteins were detected using near-IR fluorescently labeled secondary antibodies (Azure Biosystems 650 and 800; AC2165 and AC2135). Protein bands were visualized using a Sapphire Biomolecular Imager (Azure Biosystems) and analyzed with AzureSpot (Azure Biosystems).