4 to 12 nupage bis tris gels

HeLa cells were infected with 5 PFU per cell of rMVAs for 18 h, washed once with phosphate buffered saline (PBS), then lysed in LDS sample buffer with reducing agent (Thermo Fisher). The lysates were dispersed in a sonicator for four 30 s periods; the proteins were resolved on 4 to 12% NuPAGE Bis-Tris gels (Thermo Fisher) and transferred to a nitrocellulose membrane with an iBlot2 system (Thermo Fisher). The membrane was blocked with 5% nonfat milk in Tris-buffered saline (TBS) for 1 h, washed with TBS with 0.1% Tween 20 (TBST), and then incubated at 4°C overnight with rabbit anti-CoV-2 RBD polyclonal antibody (Cat# 40592-T62, Sino Biological) or anti-FLAG M2 peroxidase antibody (Cat# A8592, MilliporeSigma) in 5% nonfat milk in TBST. The membrane that had been incubated with anti-RBD antibody was then incubated for 1 h with secondary antibody conjugated to horseradish peroxidase (Jackson ImmunoResearch). After washing, the membrane bound proteins were detected with SuperSignal West Dura substrate (Thermo Fisher).

Cells were harvested, washed, and lysed in Lysis buffer (20 mM Tris (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100 and protease inhibitor) on wet ice for 30 min with frequent agitation. Cell lysates were cleared by centrifugation at 13,000 xg for 10 min at 4°C; the proteins were resolved on 4 to 12% NuPAGE Bis-Tris gels (Thermo Fisher) and transferred to a nitrocellulose membrane with an iBlot2 system (Thermo Fisher). The membrane was blocked with 5% nonfat milk in Tris-buffered saline (TBS) for 1 h, washed with TBS with 0.1% Tween 20 (TBST), and then incubated with the primary antibody in 5% nonfat milk in TBST overnight at 4°C. The membrane was washed with TBST and incubated with the secondary antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch) in TBST with 5% nonfat milk for 1 h. After the membrane was washed, the bound proteins were detected with SuperSignal West Dura substrates (Thermo Scientific).