Coomassie staining

The 293 T/ACE2 and 293 T cells were transfected with CMV-Luc-IRES-EGFP-SV40 and pCDNA3.1-SARS-S or pCDNA3.1-SARS2-S plasmids for cell–cell fusion, harvested, and lysed on ice in RIPA lysis buffer (Boston BioProducts, Ashland, MA) in the presence of cOmplete Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, MO). The protein concentrations were measured by Coomassie staining (Bio-Rad). Proteins were separated by 10% SDS-PAGE, transferred to nitrocellulose membranes, which were blocked with 5% non-fat milk, 0.1% Tween-20 in Tris-buffered saline (TBS) and incubated with anti-Myc antibody (Cell Signaling, Danvers, MA), anti-C9 antibody (Bionova, Freemont, CA) (Cell Signaling), or anti-SARS-CoV-2 S RBD antibody (Cell Signaling). After washing, the membranes were incubated with horseradish peroxide (HRP)-conjugated secondary antibodies (Cell Signaling), and the proteins were detected using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare BioSciences, Pittsburgh, PA). Protein bands were captured by Chemi Doc MP Imaging System. The membranes were stripped and re-probed with an anti-actin antibody (Cell Signaling) for a loading control.

SDS-PAGE was performed under both reducing and non-reducing conditions. The rHA samples were mixed with 2× Laemmli buffer (Bio-Rad, 161–0737), and approximately 4-5 μgs of rHA was loaded per lane. For reducing conditions, a final concentration of 100 mM DTT was added to the Laemmli-rHA solution using a 500 mM DTT stock (Pierce, product# 20291, lot# ND170603) and incubated in a 100°C heat block for 3–5 min prior to loading on to the gel. Non-reduced and reduced samples were separated using either 3-8% Tris-Acetate gels (Life Technologies, EA03752BOX) and Tris-Acetate SDS Running Buffer (Invitrogen, LA0041) or 4-12% NuPAGE Bis-Tris Gels (Cat# NP0323, Life Technologies Corporation, Carlsbad, CA) and 1× MES Running Buffer (50 mM MES, 50 mM Tris, 0.1% sodium dodecyl sulfate, 1 mM EDTA pH 7.3) at 150 volts for 1 hour. Protein bands were visualized by Coomassie staining (Bio-Rad, 161–0787). Gels were stained for 1 hour followed by a water wash until protein bands developed. Alternatively, purity and trypsin resistance gels were stained for 1 hour in 0.1% Brilliant Blue R, 7.7 M reagent alcohol, 1.75 M glacial acetic acid, and de-stained in 10% acetic acid. Gels were scanned using Gel Logic 212 Pro Imaging System, and densitometry was performed using the Carestream Molecular Imaging software (Carestream Health, Incorporated, New Haven, CT).