0.45 m pvdf membrane

A 1 mL volume of the supernatant containing rVSV-SC2 was pelleted by centrifugation at 30,000 × g for 2 h and lysed in 30 µL of NP-40 lysis buffer (Invitrogen) for 30 min on ice. Viral lysates were mixed with 4× Laemlli buffer (Bio-Rad) supplemented with 10% β-mercaptoethanol and denaturated at 95°C for 5 min. Proteins were separated by SDS-PAGE on a 4%–20% Mini-PROTEAN TGX Gel (Bio-Rad) and transferred onto a 0.45-µm PVDF membrane (Thermo Scientific). Membranes were blocked for 1 h at room temperature in TBS-T (20 mM tris, 150 mM NaCl, 0.1% Tween-20, pH 7.5) supplemented with 3% bovine serum albumin (BSA; Sigma). Membranes were then incubated for 1 h at RT with the following primary antibodies: mouse anti-SARS-CoV-2 S2 (dilution 1:2,000, clone 1A9, GeneTex) and mouse anti-VSV-M (dilution 1:1,000, clone 23H12, Kerafast). Membranes were washed three times with TBS-T and incubated 1 h at RT with an HRP-conjugated anti-mouse secondary antibody (dilution 1:50,000, G-21040, Invitrogen). After washing three times in TBS-T, the signal was revealed with SuperSignal West Pico PLUS (Thermo Scientific) following the manufacturer’s instructions. Images were acquired on an ImageQuant LAS 500 (GE Healthcare) and analyzed with Fiji software.

Protein samples were prepared by lysing cell pellets in RIPA buffer (150 mM NaCl, 1% v/v Igepal C630 NP-40, 0.5% w/v sodium deoxycholate, 0.1% w/v SDS, 5 mM EDTA, 50 mM Tris HCl pH 8.0, and protease/phosphatase inhibitors), followed by sonication (2.5 W, 10 s), and heat-denatured in SDS-loading buffer (50 mM Tris HCl pH 6.8, 2% w/v SDS, 10% v/v glycerol, 1% 2-mercaptoethanol, 0.01% w/v bromophenol blue) for 5 min at 95 °C. Protein concentration was measured using a BCA Protein Assay Kit (Pierce, Thermo Fisher Scientific). 20 µg of total protein per sample was resolved by SDS-PAGE, electrophoretically transferred onto 0.45 µm PVDF membrane (Thermo Fisher Scientific) and blocked with 5% w/v skim milk powder in TBST buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.1% v/v Tween 20). Proteins of interest were labeled overnight at 4 °C with primary antibodies in milk/TBST. Membranes were washed three times in milk/TBST for 10 min, incubated with HRP-conjugated secondary antibodies (BioRad) for 1 h, and again washed three times in milk/TBST. SuperSignal West Pico Plus Chemiluminescence Substrate (Pierce) was used for detection and digital images were captured using an ImageQuant LAS 4000 (GE Healthcare Life Sciences).

Cells were harvested and resuspended in RIPA buffer (Alfa Aesar, Haverhill, MA, USA) supplemented with protease and phosphatase inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) and incubated in ice for 60 min. Lysed cells were centrifuged at 15,000 rcf for 20 min at 4 °C and the supernatant was isolated. Protein concentrations were determined using a bicinchoninic acid assay (Thermo Fisher Scientific, Waltham, MA, USA).
Proteins were separated by a 12% SDS/PAGE gel, transferred onto a 0.45 µm PVDF membrane (Thermo Fisher Scientific, Waltham, MA, USA), and probed with the appropriate antibodies. Chemiluminescence signal was obtained using ECL^TM Prime (Cytiva, Marlborough, MA, USA), and blot images were captured using the ChemiDoc^TM Imaging System (Bio-Rad, Hercules, CA, USA).