SDS-PAGE and Western Blotting Protocol

SDS-PAGE and Western blotting were performed according to standard protocols [33 (link)]. Briefly, protein samples were separated on 12% SDS-PAGE gels and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Billica, USA). Membranes were blocked with 5% (w/v) nonfat milk in PBS containing 0.05% Tween (PBST) for 1 h at 37 °C. Rinsed blots were incubated with anti-His monoclonal antibody (1:5000 in PBS, Abcam, Cambridge, UK), CD163, or PRRSV-specific monoclonal antibodies (prepared in our lab) at 37 °C for 1 h (h) respectively, and with HRP-conjugated goat anti-mouse IgG (1:5000 in PBS, Sungene, Shanghai, China) at 37 °C for 1 h. Signals were detected by SuperSignal West Pico/Femto Chemiluminescent Substrate (Thermo Scientific, MA, USA) and images were captured with Gel 3100 chemiluminescent imaging system (Sage Creation Science, Beijing, China).