To examine the expression of viral proteins and firefly luciferase protein, we infected PK-15 and ST cells with rPRV HN1201-EGFP-luc or PRV HN1201 at a multiplicity of infection (MOI) of 0.1 for 24 h. Cells were collected in lysis buffer (50 mM Tris–HCl, pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 2 mM MgCl2) supplemented with protease and phosphatase inhibitor cocktail (HY-K0010 and HY-K0022, MedChemExpress). The protein concentrations in the lysates were quantified with a BCA Protein Assay Kit (DingGuo, Beijing, China) and detected with a microplate reader (Awareness Technology, Inc., Palm City, FL, USA). Protein samples (30 μg) were separated by SDS-PAGE and transferred to nitrocellulose membranes (ISEQ00010, Millipore), which were incubated in 5% nonfat milk (A600669, Sangon) for 1 h at room temperature. The membrane was incubated with anti-firefly luciferase antibody (1:3000) (ab185924, Abcam), rabbit antiserum against PRV gE (1:500) (generated by immunizing a rabbit with purified recombinant gE), anti-PRV gB mouse monoclonal antibody (1:2000) (prepared and stored by our laboratory) or anti-β-actin antibody (1:5000) (A1978, Sigma-Aldrich) at 4 °C overnight and then incubated with horseradish peroxidase (HRP)-conjugated secondary antibody (715-035-150 or 711-035-152, Jackson ImmunoResearch Laboratories) for 1 h. Immunoblotting results were visualized with Luminata Crescendo Western HRP Substrate (WBLUR0500, Millipore) on a GE AI600 imaging system.

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