Bca method

Cells and tissues were collected and lysed in cell/tissue lysis buffer (Solarbio, Beijing, China). Proteins were extracted, and the protein concentration was measured by the BCA method (Solarbio, Beijing, China). Proteins were separated by 10% sodium dodecyl sulfate (SDS)-PAGE and transferred to 0.45-μm polyvinylidene difluoride membranes (Invitrogen, CA, USA), which were blocked with blocking buffer (EpiZyme, Shanghai, China), incubated overnight with primary antibodies, and washed and incubated with the HRP-conjugated anti-rabbit secondary antibody (Abcam, UK) for 1 h. Peroxidase activity was visualized via the ECL method (EpiZyme, Shanghai, China).

Proteins were extracted from left ventricular myocardium with RiPA Buffer (Thermo Scientific) and protease inhibitor was added. Protein concentration was determined by BCA method (Solarbio, Beijing, China). Proteins were taken at 50 μg and denatured at 100°C for 5 mins. Proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China) and then transferred to a polyvinylidene fluoride (PVDF) membrane by electrophoretic equipment (Bio-Rad, CA, USA). After being blocked with 3% BSA for 2 hours at room temperature, the membranes were incubated with anti-PI3K antibody, anti-phospho-PI3K antibody, anti-eNOS antibody, anti-phospho-eNOS (1 : 1000, Bioss, Beijing, China), anti-COX-2 antibody, anti-HMGB-1 antibody (1 : 1000, Proteintech, Chicago, IL, USA), and anti-ECE-1 antibody (1 : 2000, Signalway Antibody, College Park, MD, USA), primary antibodies overnight at 4°C. Next day, after being washed with TBST for 3 times, the membranes were incubated with HRP-labeled goat anti-rabbit IgG (1 : 25000, 1 h, Zhongshanjinqiao, Shanghai, China) at room temperature. After being washed with TBST for 3 times, the membranes were visualized by Pierce™ ECL Western Blotting Substrate and imaged using FluorChem E Imaging System (Protein Simple, USA), which were normalized to β-actin as an internal control.

Total protein was isolated using a total protein isolation kit (Solarbio, China) and its concentrations were estimated with the BCA method (Solarbio, China). Proteins (30 μg/per well) were applied for electrophoresis (5%∼10% SDS-PAGE) and then transferred onto polyvinylidene difluoride membranes (0.2 µM, Bio-Rad, USA). Subsequently, the PVDF membrane was blocked for 1.5 h at room temperature and then incubated with primary antibodies against iNOS (1:1000, ab178945), Arg1 (1:1000, CST93668), SIRT1 (1:1000, ab189494), acetyl-NF-κB p65 (Lys310) (1:1000, CST3045), phospho-NF-κB p65 (Ser536) (1:1000, CST3033), NF-κB-p65 (1:1000, CST8242), β-actin (1:2000, ab8226) overnight at 4 °C, and then incubated with secondary antibodies (1:3000, CST7074) for 1 h. Enhanced chemiluminescence (170-5070, Bio-Rad, USA) was applied for blots developing and the density was calculated by ImageJ system.

DEF cells in 6-well plates, which were separately infected with the parental virus and mutant viruses, were collected at various time points post-infection. Cells were lysed with Cell Lysis Buffer for Western and IP (IP: immunoprecipitation; Beyotime, Shanghai, China). Protein concentrations were determined by the BCA method (Solarbio, Beijing, China). Equal protein amounts were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred from the gel to polyvinylidene difluoride membranes by wet transfer. The membranes were blocked with 5% skimmed milk in PBST (PBS/Tween) at 37 °C for 2 h. The membranes were incubated at 37 °C for 1 h with anti-E protein mAb or anti-β-actin mAb (1:5000 dilution in PBST). After three washes with PBST, the membranes were incubated with secondary antibodies for 1 h at 37 °C. Finally, the protein bands were detected using the BeyoECL Plus Kit after three washes (Beyotime, Shanghai, China).