Horseradish peroxidase hrp labeled goat anti mouse igg antibody

EH was purchased from Roche (China). Heparin was purchased from Tianjin Biochemical Pharmaceutical Company. Anti-Bcl-2 antibody, anti-Bax antibody, anti-Caspase-3 antibodies, horseradish peroxidase (HRP)-labeled goat anti-mouse IgG antibody, and HRP-labeled goat anti-rabbit IgG antibody were purchased from Abcam (USA). Cleaved Caspase-3 antibody was purchased from CST/Cyxnet (USA). Mouse tumor necrosis factor (TNF)-α ELISA kit and TransStart Top Green qPCR Supermix were purchased from Beijing TransGen Biotech. The mouse histone H4 ELISA kit, mouse NAGL ELISA kit, and mouse KIM-1 ELISA kits were purchased from Shanghai Guangrui Biotech. Purified anti-β-actin and the fluorescein isothiocyanate (FITC)–annexin V apoptosis detection kit with 7-amino-actinomycin D (7-AAD) were purchased from Biolegend (USA). Fetal bovine serum was purchased from GIBCO (USA). DCFH-DA probe was purchased from Shanghai Beyotime Biotechnology.

P3 rAd-PEDV-S was used to infect HEK293A cells, and the cells were harvested after 120 hours. The cells were lysed using the lysis buffer (Thermo Scientific, Waltham, MA) containing protease inhibitors (Invitrogen, USA), frozen/thawed 3 times, and then centrifuged and collected at 4°C and 12000 rpm for 10 minutes. Then, the proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes under a constant voltage of 80 V for 2 hours. The blots were blocked with 5% skim milk at 37°C for 1 hour and then incubated with a primary mouse anti-His-tag monoclonal antibody (mAb) (1 : 1000 dilution; Abcam, USA) for 1 hour at 37°C. After washing 3 times for 5 minutes per wash with TBS-Tween 20 (50 mM Tris, 150 mM NaCl, and 0.05% Tween 20, pH 7.6), the membrane was incubated with a secondary horseradish peroxidase- (HRP-) labeled goat anti-mouse IgG antibody (1 : 2000 dilution; Abcam, USA) for 1 hour at 37°C. Finally, the membrane was washed and visualized using an ECL chemiluminescent substrate reagent kit (Thermo Scientific, USA).