Ice cold lysis buffer

Protein homogenates from goat PBMCs were extracted as previously described (27 (link)). Briefly, the cells were lysed for 20 min on ice in ice-cold lysis buffer (Roche). The lysates were centrifuged at 12,000 × g for 20 min at 4°C to obtain a clear lysate. The protein content of each sample was determined using the BCA Protein Assay Kit (Thermo Scientific). Then, equal amounts of protein were separated on a 12% SDS-polyacrylamide gel and transferred to polyvinylidene difluoride membranes. Membranes were probed overnight at 4°C with an anti-PPRV-N monoclonal antibody provided by China Animal Health and Epidemiology Center (Qingdao, China), or a rabbit polyclonal antibody against sheep SLAM (1:2000; Santa Cruz). The bands were visualized using horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:15000, Boster) or goat anti-rabbit IgG (1:20000, Boster) prior to the ECL protocol (Amersham Biosciences, Piscataway, NJ, USA). As an internal standard, all membranes stripped with primary antibodies were reprobed with anti-β-actin antibody (Invitrogen). Changes in protein expression were determined after normalizing the band intensity of each lane to that of β-actin. Signal was visualized using the Konica SRX 101A developer (Konica Minolta Medical Imaging, Wayne, NJ, USA) and the Quantity One software (Bio-Rad, Mississauga, ON, Canada) was used to densitometrical analysis.