Bovine serum

The human cell lines: DU145 (DSMZ ACC 261), MDAMB231 (DSMZ ACC 732), Panc-1 (DSMZ ACC 783) and hTERT RPE1 (ATCC CRL-4000) were grown in Dulbecco’s MEM medium supplemented with 10% fetal bovine serum. SHYS5Y (DSMZ ACC 209), HeLa (DSMZ ACC 57) and MCF-7 (DSMZ ACC 115) were grown in RPMI medium supplemented with 10% fetal bovine serum, MDAMB435S (ATCC HTB-129) were grown in RPMI medium supplemented with 15% fetal bovine serum and MiaPaca-2 (DSMZ ACC 733) cells were grown in DMEM-F12 medium supplemented with 10% fetal bovine serum. All media were purchased from Thermo Fisher Scientific (Schwerte, Germany), and bovine serum from Biochrom (Berlin, Germany) or PAA (GE Healthcare, Freiburg, Germany). All cell lines were incubated under a humidified atmosphere of 95% air/5% CO₂ at 37 °C.

BVDV-1a NADL strain (ATCC VR-534), Bovine rotavirus (BRV) strain NCDV (ATCC VR-452) Pasteurella multocida P-1059 (capsular serogroup A) were obtained from China Veterinary Culture Collection Center (CVCC). BoHV-1 isolate HLJ07, BRSV field isolate HLJ01, Bovine coronavirus (BCoV) isolate HM and Mannheimia haemolytica SH1801 (Serotype 1), and BPIV-3 (HQ530153) were isolated by our laboratory (23 (link)). BVDV-2 HLJ-10 strain was provided by Dr. Mingchun Gao at Northeast Agricultural University (24 (link)). The Nucleic Acid of CSFV vaccine strain (C-strain) was provided by Dr. HuaJi Qiu at Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences (25 (link)). BVDV, BoHV-1, BPIV3 were propagated on Madin-Darby bovine kidney (MDBK) cells. BRSV was propagated on bovine Turbinate (BT) cells cultured in Minimum Essential Medium (MEM) (Thermo Fisher, Inc.; USA) supplemented with 2% bovine serum (GE, Inc.; USA). IDV strain D/bovine/Mississippi/C00046N/2014 was obtained via reverse genetics system and propagated on Madin-Darby Canine Kidney (MDCK) cells (26 (link)).

SDS-PAGE was carried out according to the procedure of Laemmli with some modifications [22 (link)]. Briefly, the protein samples were prepared by mixing with 1x final concentration of loading sample buffer (0.1 M tris-HCl, pH 6.8, 6% sodium dodecyl sulphate, 30% Glycerol, 15% 2-mercaptoethanol and 0.01% bromophenol blue). Prior to loading to the gel, the samples were heated in a boiling water bath for 5 min. The discontinuous gel system usually had 5% stacking and 12.5% resolving gel. Electrophoresis was carried out using Laemmli buffer at constant current of 15 mA first, till the samples entered into the resolving gel and then at 20 mA till the completion. On completion of electrophoresis, gel was immersed in 0.05% Coomassie Blue R250 in methanol: acetic acid: water (4:1:5) with gentle shaking and was then destained in destaining solution (staining solution without dye) till the background was clear. The protein standards (GE Healthcare, UK) used were phosphorylase b, rabbit muscle (97,000 Da), albumin, bovine serum (66,000 Da), ovalbumin, chicken egg white (45,000 Da), carbonic anhydrase, bovine erythrocyte (30,000 Da), trypsin inhibitor, soybean (20,100 Da), and α lactalbumin, bovine milk (14,400 Da).