Western Blot Analysis of PEDV N Protein

Cells were lysed using cell lysis buffer (ThermoFisher Scientific, China) containing the protease inhibitor PMSF to prevent protein degradation. After measuring the protein concentration, 5× protein buffer was added and the proteins were denatured in a 98°C water bath. The protein samples were separated on 12% SDS–PAGE Gel SuperQuick Preparation Kit (Beyotime, China), alongside the prestained color protein marker (ThermoFisher Scientific, China). The proteins on the gel were transferred to PVDF membranes (Millipore, China) using a Trans-blot Turbo (Bio-Rad, China), and then blocked with QuickBlock™ Western Blocking Buffer (Beyotime, China). Membranes were cut, based on the bands of the protein marker, immediately after blocking. The excised membranes were incubated overnight at 4°C with PEDV N antibody (Medgene Labs, Brookings, SD, USA) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; ABclonal, China). The corresponding HRP-conjugated goat anti-mouse and anti-rabbit IgG (H + L) secondary antibodies were added the next day, followed by incubation for 1 h at room temperature. After elution, imaging was performed on a Bio-Rad imager with diluted NcmECL Ultra (NCM, China).