Viral Overlay Protein Binding Assay

The membrane proteins from BHK-21 cells were isolated using Mem-PER Plus Kit (Thermo) in accordance with the manufacturer’s protocol. VOPBA was performed as essentially described in Thepparit and Smith (2004). Briefly, membrane proteins were subjected to electrophoresis through 12% SDS-PAGE and transferred to PVDF membranes. The membrane containing transferred proteins was blocked with 5% BSA in PBST at 37°C for 2 h. For viral overlay, the membranes were incubated with 10⁵ TCID₅₀ of TMUV in 5% BSA in PBST overnight at 4°C and washed three times with PBST buffer. Subsequently, the membranes were incubated with monoclonal antibody against TMUV at 37°C for 1 h followed by incubated with a alkaline phosphatase-conjugated goat anti-mouse IgG. Finally, the signal was developed using Alkaline Phosphatase Assay Kit (Beyotime) in accordance with the manufacturer’s protocol.