Anti caspase 3

Cells were washed twice in PBS and lysed in RIPA buffer (Solarbio, China) with 1 mM phenylmethanesulfonyl fluoride (PMSF). A BCA protein kit was used to quantify protein concentrations (Beyotime, China). Total proteins (30 μg) were separated on 15% SDS-PAGE gels and transferred to polyvinylidene difluoride (PVDF) membranes (Beyotime, China). The membranes were then blocked in TBST with 3% non-fat milk (Sangon Biotech, China) at 25°C for 2 h and incubated with anti-survivin (1:1,000), anti-caspase-3 (1:1,000), anti-Bcl-2 (1:3,000), anti-P-gp (1:3,000), and anti-β-actin (1:5,000) primary antibodies (Proteintech, USA) overnight at 4°C. Next, the membranes were incubated with the appropriate HRP-conjugated secondary antibodies (1:10,000) (Proteintech, USA) at 25°C for 1 h. Protein bands were visualized by ECL chemiluminescence kit (Sangon Biotech, China), and the gray values of the protein bands were analyzed by Image J.

VERO (ATCC®CRL-1586) and PK-15 cells (ATCC®CCL-33) were cultured in Dulbecco’s modified Eagle’s medium (CORNING, USA) supplemented with 10% fetal bovine serum (Gibco) at 37°C in a humidified atmosphere containing 5% CO2. The PRV strain ZJ 01 was isolated from a pig herd at China in 2012 (Gu et al., 2015 (link)), which was used for all experiments. The PRV were proliferated in PK-15 cells and stored at −80°C. β-Streptococcus equinus strain SheepZ001 were cultured in Tryptone soybean broth (TSB) + 0.05% fetal bovine serum (FBS) medium at 37°C, 220 rpm. Pasteurella multocida HB01 (Serotype A) were cultured in Brain-heart extract broth (BHI) medium at 37°C, 220 rpm. Bacillus subtilis 168, WB800 and other Bacillus subtilis group isolated from sheep nasal cavity were cultured in Luria-Bertani (LB) medium at 37°C, 220 rpm. Anti-PRV gB-protein monoclonal antibodies (1B1, prepared and stored in our laboratory), anti-GAPDH antibody (Proteintech), anti-cytokeratin18 (Abcam), anti-claudin 1(Abcam), anti-caspase3 (Proteintech); anti-caspase9 (Proteintech); anti-ISG15 antibody (Abcam), anti-PCNA antibody (Abcam), anti-CD3 antibody (Dako), anti-IgA antibody (Abcam) and anti-CD208 antibody (Dendritics) was used in the study.

Protein levels in mouse tissues or cells were determined by western blotting using the following antibodies: anti‐TPPP2 (Abcam, 121215; 1:1000), anti‐green fluorescent protein (GFP) (EnoGene, E12‐026‐4; 1:3000), anti‐β‐ACTIN (Merck Millipore, MAB1501; 1:7500), anti‐Caspase3 (Proteintech, 19677‐1‐AP; 1:500), anti‐Bax (Proteintech, 50599‐2‐Ig; 1:500), anti‐Bcl‐2 (Proteintech, 12789‐1‐AP; 1:500) and anti‐Eef1b (Proteintech, 10483‐1‐P; 1:1000).

Western blot assays were performed as previously described [29 (link)]. The primary antibodies used in this study were anti-SOX9 (#A2479, ABclonal, Wuhan, China; 1:2000), anti-PCNA (#13110, Cell Signaling Technology, Shanghai, China; 1:1000), anti-Ki67 (#ab16667, Abcam, Shanghai, China; 1:1000), anti-CDK2 (#ab232753, Abcam, Shanghai, China; 1:1000), anti-CYP11A1 (#D122183, Sangon, Shanghai, China; 1:1000), anti-CYP19A1 (#A2161, ABclonal, Wuhan, China; 1:1000), anti-StAR (#8449S, Cell Signaling Technology, Shanghai, China; 1:1000), anti-caspase3 (#19677-1-AP, Proteintech, Wuhan, China; 1:1000), anti-GAPDH (#TA802519, ORIGENE, Wuxi, China; 1:3000), and anti-β-tubulin (#AC008, ABclonal, Wuhan, China; 1:3000). The corresponding HRP-conjugated secondary antibodies obtained from Sangon Biotech (Shanghai, China) were diluted in 0.25% BSA/TBST solution. The protein levels of β-tubulin and GAPDH served as internal controls in oxidative and non-oxidative experiments, respectively. Each group had three independent biological replicates. The replicates with the highest representativeness were selected and are shown in the figures, and their corresponding normalized fold change values were calculated and are listed under the blot images.

McCoy's 5A medium, RPMI-1640, 100 U/mL streptomycin, 100 μg/mL penicillin and PBS were acquired from gibco (Life Technologies, USA). Fetal bovine serum was obtained from BI (Bioind, Israel). SRB assay kit was obtained from BestBio (Shanghai, China). Cell cycle and apoptosis analysis kits were purchased from Yeasen Biotech (Shanghai, China). Annexin V-FITC/PI apoptosis kit was obtained from Multisciences (Hangzhou, China). Reactive Oxygen Species Assay Kit was obtained from Beyotime (Shanghai, China). Anti-PARP1, anti-caspase 3, anti-GAPDH, anti-GSK3β, anti-p-GSK3β (Ser9), anti-Cyclin D1, anti-β-actin, anti-PI3K, anti-Akt, and anti-p-Akt (Ser473) antibodies were purchased from ProteinTech (Wuhan, China). Anti-cleaved caspase 3 was purchased from Cell Signaling Technology (Beverly, MA, USA). Ultrapure water was produced by the ultrapure water system (Yipu Yida Technology, Nanjing, China). Cisplatin was purchased from Acmec (Shanghai, China). All the analytically pure chemicals and solvents were purchased from Sinopharm Group (Shanghai, China).

The protein levels of YAP1, P-YAP, TAZ, LATS1, Claudin-1, and Caspase 3 were evaluated by western blotting. Total protein was extracted from the colon mucosa with RIPA buffer (Beyotime Biotechnology, Shanghai, China) supplemented with protease and phosphatase inhibitors (Epizyme Biotechnology Co., Ltd, Shanghai, China). Then, the proteins were separated and transferred to polyvinylidene difluoride membranes, which were probed with anti-YAP1 monoclonal antibody (1 : 1000), anti-P-YAP antibody (1 : 1000), anti-TAZ monoclonal antibody (1 : 1000), anti-LATS1 monoclonal antibody (1 : 1000), anti-Claudin-1 monoclonal antibody (1 : 1000), and anti-Caspase 3 (1 : 1000, Proteintech Group, Inc., Wuhan, China), respectively. The above primary antibodies were bought from Cell Signaling Technology (USA). Protein bands after overnight incubation with the primary antibodies were incubated with an HRP-labelled antibody (1 : 10000, Epizyme Biotechnology Co., Ltd, Shanghai, China). The results were detected with chemiluminescence detection reagents (Meilun, Dalian, China) following the manufacturer's instructions.