Ripa lysate

Proteins were lysed by RIPA lysates (Solarbio, Beijing, China) added with PMSF (Solarbio, Beijing, China) and phosphatase inhibitors (PhosphoStop, Roche, Germany). And then proteins were separated and transferred to PVDF membranes. Membranes were incubated overnight with the following primary antibodies: PGAM5 (1:500, cat.no.sc515880,santa cruz, CA, USA), Tom20(1:8000, cat.no. 11802-1-AP, Proteintech, Wuhan, China), MFF (1:20,000, cat.no. 17090-1-AP, Proteintech, Wuhan, China), Fis1 (1:1000, cat.no. 10956-1-AP, Proteintech, Wuhan, China), MFN2 (1:20,000, cat.no. 12186-1-AP, Proteintech, Wuhan, China), β-actin (1:20,000, cat.no. 20536-1-AP, Proteintech, Wuhan, China), β-tubulin (1:10,000, cat.no. 10094-1-AP, Proteintech, Wuhan, China), and p-Drp1 (1:1000, cat.no. 3455, CST, USA). HRP-labeled secondary antibody was applied at room temperature for 1 h. The chemiluminescence signal was detected using an Amersham Imager 800 (Amersham Biosciences, Buckinghamshire, UK).

RIPA lysate (Solarbio) and protease inhibitor PMSF (Solarbio) with a final concentration of 1 mM were added into the cells, and then, the total protein of the cells was extracted by centrifugation. The total protein concentration was tested by the BCA method, the final loading protein concentration was adjusted to 5–10 μg/μl, the sample was loaded, and 10% SDS-PAGE separation gel was applied for gel electrophoresis. The target protein was transferred to PVDF membrane by the semiwet transfer method, sealed with 5% skimmed milk powder for 1 h, and then added with SIRT1 (60303-1-Ig, Proteintech, 1 : 4000) and MMP9 (10375-2-AP, Proteintech, 1 : 1000) primary antibody diluent at 4°C overnight. After being washed with 0.1% PBST buffer for 3 times, the corresponding HRP-labeled secondary antibody (SA00001-1/2, Proteintech, 1 : 5000) was added and incubated at room temperature for 1 h and then washed with 0.1% PBST buffer for 3 times. ECL chemiluminescence solution (Solarbio) was evenly dripped on PVDF membrane, and the expression of protein was analyzed by ImageJ.

Hippocampal or cell proteins were extracted using RIPA lysate (containing protease inhibitors, Solarbio, China). Samples were completely disrupted at 4 °C for 30 min, followed by centrifugation at 13,000 g for 15 min, and the determination of total protein concentration by bicinchoninic acid assay (BCA, Solarbio). After density at 95°C for 10 min, 30 μg of protein was loaded onto an SDS-PAGE gel for subsequent electrophoresis. Western blotting was performed as previously described 11 (link), 41 (link), 42 (link). ChemiDoc XRS (Bio-Rad, USA) was used to visualise immunoreactive proteins. The relative intensity of the bands was quantified using the Image Lab Analyzer software (Bio-Rad). The antibodies used in this study are listed in Table 1.

RIPA lysate (Solarbio, Beijing, China) containing protease inhibitors was used to extract IPEC-J2 cells protein. The BCA Protein Concentration Assay Kit (Beyotime) was used to calculate the protein concentration. Following a 10 min incubation at 100 °C, the protein underwent denature. Electrophoresis using 10% SDS-PAGE was then carried out. Membranes made from polyvinylidene difluoride (PVDF) were used for transferring the proteins. The membranes were blocked with 5% skimmed milk for 2 h at room temperature after being rinsed once with tris-buffered saline with Tween (TBST). The primary antibodies (Bioss, Beijing, China) were then added and diluted, and incubated at 4 °C overnight. The membranes were washed with TBST for five minutes the next day, and the procedure was repeated three times. The membranes were subjected to diluted secondary antibody (IgG/HRP) incubation at room temperature for 1.5 h, followed by three TBST washes. Eventually, the enhanced chemiluminescence (ECL) kit (Proandy, Xian, China) was used to observe protein bands. Table S2 provides specific information of antibodies.

JEG-3 cells and mouse placenta tissues with HEV infection were treated with RIPA lysate (Solarbio, Beijing, China) and lysed for 20 min on ice. BCA protein quantification kit, which was provided by Thermo Fisher Scientific (Waltham, USA), was applied to measure and adjust the protein concentration of the samples in each group. The sample was heated with boiling water for 10 min after loading buffer (Cell Signaling Technology, Boston, USA) was added. Protein samples were separated by SDS‒PAGE (10% Bis-Tris Gel) and transferred to polyvinylidene fluoride (PVDF) membranes, which were obtained from Millipore (Bedford, USA). The membrane was transferred at a constant current of 200 mA for 50‒100 min, depending on the size of the target protein. After washing, the PVDF membranes were immersed in PBST containing 5% skim milk powder (Mengniu, Inner Mongolia, China) for 60 min at room temperature and then incubated with primary antibodies at a dilution of 1:1000 at 4°C overnight. The next day, after washing with PBST for three times, the PVDF membranes were incubated with goat anti-rabbit or mouse IgG (HRP) for 50 min at room temperature. Washing with PBST again, the PVDF membranes were developed with immobilon classico western HRP substrate (Millipore, Bedford, USA) and photographed by Tanon 5200 (Shanghai, China).