Hrp conjugated anti mouse or rabbit igg antibodies

Vero cells were seated on a 12-well plate at a cell density of 2 × 10⁵ cells/well. Cells were infected with SARS-CoV-2 at an MOI of 1 and incubated at 37°C for 24 h. The cultured cells were subjected to centrifugation at 1,600 × g for 5 min. The supernatant was removed, and the cells were lysed using 1 × SDS sample buffer. The lysed cells were boiled at 95°C for 3 min, sonicated using Bioruptor II (BM Equipment, Tokyo, Japan), and centrifuged at 16,400 × g for 3 min. The samples were loaded onto a 10% TGX FastCast Acrylamide gel (Bio-Rad, CA, USA) and electrophoresed. Then, the proteins were transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with PBS containing 0.05% tween-20 (PBS-T; Nacalai Tesque) and 5% skimmed milk (Nacalai Tesque) and reacted with rabbit anti-SARS-CoV-2 S1 (clone: SA39; Cell Engineering Corporation, Osaka, Japan) or mouse anti-S2 monoclonal antibodies (GeneTex, CA, USA). The membrane was washed with PBS-T twice and reacted with HRP-conjugated anti-mouse or rabbit IgG antibodies (Sigma-Aldrich). Protein bands were visualized using Chemi-Lumi One Ultra (Nacalai Tesque) and Amersham ImageQuant 800 (GE Healthcare, IL, USA).