Samples in SDS solubilizer [0.0625 M Tris·HCl (pH 6.8), 10% glycerol, 0.01% bromophenol blue, 2% (wt/vol) SDS, +/- 2% 2-mercaptoethanol] were heated at 95°C for 5 min, electrophoresed through 8% or 10% (wt/vol) polyacrylamide-SDS gels, transferred to nitrocellulose membranes (Bio-Rad), and incubated with rabbit polyclonal anti-SARS-CoV-2-S1 (SinoBiological, cat: 40591-T62), rabbit polyclonal anti-SARS-S2 (#JH50520001, obtained from Dr. Carolyn Machamer, Johns Hopkins University), mouse monoclonal anti-SARS-S2 (ThermoFisher, cat: MA5-35946, conjugated to HRP), goat anti-human IgG (sc-2453, Santa Cruz Biotechnologies), rabbit monoclonal anti-hACE2 (Invitrogen, cat: MA5-32307), or purified LgBiT-substrate cocktail (Promega). After incubation with appropriate HRP-tagged secondary antibodies and chemiluminescent substrate (Thermo Fisher), the blots were imaged and processed with a FlourChem E (Protein Simple).
Recombinant 4A8 was synthesized by using its published VH and VL sequences (Chi et al., 2020 (link)) to replace their counterparts in pVITRO1-M80-F2-IgG1/κ (a gift from Andrew Beavil, Addgene plasmid # 50383;http://n2t.net/addgene:50383 ; RRID:Addgene_50383, (Dodev et al., 2014 (link))). TRES328 (Peter et al., 2021 (link)) was obtained from Dr. Hans-Martin Jäck, Friedrich-Alexander-Universität.
Recombinant 4A8 was synthesized by using its published VH and VL sequences (Chi et al., 2020 (link)) to replace their counterparts in pVITRO1-M80-F2-IgG1/κ (a gift from Andrew Beavil, Addgene plasmid # 50383;