Caspase 8

Experimental monolayers were washed with serum-free media, and then total and fractionated proteins were extracted by cell lysis buffer (Cell Signaling Technology, Danvers, MA). The lysates were centrifuged at 12,000 g for 20 min at 4°C. An equal amount of protein after concentration was determined by the Bradford assay (Bio-Rad, Hercules, CA) was loaded on SDS-PAGE and transferred to nitrocellulose membranes (Bio-Rad). After blocking, specific antibodies such as ADRP, FOXO3a, caspase-8, and β-actin from ABclonal Biotechnology (Wuhan, China); Perilipin-1 from Cell Signaling Technology (Danvers, MA); and vimentin from Abcam (Cambridge, MA) were used to perform detection. Finally, each protein was detected using an enhanced chemiluminescence system (GE Healthcare, Chicago, IL, USA). Blot images were digitized (Chemidoc, Bio-Rad, Milan, Italy), and the area of each band was quantified using the computerized imaging system (QuantityOne, Bio-Rad). Relative OD (arbitrary units) was normalized for control bands in each series and for protein loading (as probed by anti-actin blots). Each test was performed in triplicate experiments.

The purified BoHV-1 gD protein (20 μg) or BoHV-1 (20 μg) was separated through sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore Schwalbach, Germany). Next, the membrane was blocked with 5% bovine skimmed milk and then incubated with BoScFv-PE38 labeled with horseradish peroxidase (HRP) at 37°C for 1.5 h. Thereafter, the membrane was washed three times with PBS containing 0.5% Tween-20. The membrane was finally developed with ECL solution (ThermoFisher Scientific, United States) and visualized with the Odyssey Infrared Imaging System (LI-COR Biosciences).
The expression of apoptotic proteins in BoHV-1-infected MDBK cells after treatment with BoScFv-PE38 was analyzed as described by Decker et al. (2004) (link). Fifty micrograms of protein was separated via 12% SDS-PAGE and transferred to PVDF membranes. The samples were subsequently hybridized with antibodies against PARP-1, Bcl-2, Bid, caspase-3, caspase-8, caspase-9 and β-actin (ABclonal Biotech Co., Ltd, China). Blots were developed using Super Signal chemoluminescent substrates (Pierce, KMF GmbH, St. Augustin, Germany).

The pET28a expression system was obtained from GE Healthcare; the pEGFP-N1 vector was obtained from Clontech; The DNA encoding for segment from 259 amino acids to 345 amino acids of glycoprotein D (AFB76672.1) was amplified by polymerase chain reaction with the primers as following: Sense primer: 5′-GAATTCATGGAGGAGTCGAAGGGC-3′ and anti-sense primer:5′-CTCGAGGATGGCTTCGAGGCTCG-3′, and the DNA fragment was cloning into the pEGFP-N1 vector for construction of the pEGFP-N1-gD, which was used to efficiently express green fluorescent protein (GFP) fused gD protein in 293T cell. Calf antiserum against BoHV-1 was from China Veterinary Culture Collection Center. A mouse anti-His monoclonal antibody (McAb), Alexa Fluor 555- conjugated anti-His antibody, FITC-labeled goat anti-mouse antibody and TRITC-labeled goat anti-mouse antibody were purchased from ThermoFisher Scientific (United States), and a FITC-labeled rabbit anti-bovine antibody was purchased from BioVision (United States). The antibodies against PARP-1, Bcl-2, Bid, caspase-3, caspase-8, caspase-9 and β-actin were purchased from ABclonal Biotech (China). The BoScFv-PE38 was labeled with horseradish peroxidase (HRP) by Sangon Biotech Co., Ltd. (China).