Tanon 5200

Cells (washed three times with PBS) and the tissues were lysed in a RIPA lysis buffer (CW2333, CWBIO, China) with protease inhibitor (CW2200, CWBIO, China) and phosphatase inhibitor cocktail (CW2383, CWBIO, China) for 45 min. Protein concentrations of cell lysates and tissue lysates were determined using a Pierce BCA Protein Assay Kit (CW0014, CWBIO, China) according to the manufacturer's instructions. Total protein (30-50 μg) for each sample was separated by the SDS–PAGE and transferred to polyvinylidene difluoride (PVDF, Millipore, IPVH00010) membranes. The membranes were blocked with 5% nonfat milk at room temperature for 2 h and then incubated with primary antibodies overnight at 4°C (for all antibodies, see Supplementary Table2). After washing, the membranes were incubated with a 1 : 8,000 dilution of goat anti-mouse or anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (CW0102 and CW0103, CWBIO, China) at room temperature for 1.5 h. Protein bands were visualized using an Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500, Millipore) via an automatic chemiluminescence imaging analysis system (Tanon-5200, USA). ImageJ (NIH, National Institutes of Health, USA) was used to analyse these images.

Liver tissues or cells were homogenized with RIPA lysis buffer and centrifuged at 12,000 rpm for 15 minutes before the supernatant was collected. Protein concentration was determined using the BCA protein assay kit (ComWin Biotech, Beijing). After 30 µg proteins were denatured by placing samples in an environment at 95°C for 5 minutes, proteins were then separated through 10% acrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Darmstadt, Germany). The membranes were incubated with primary antibodies at 4°C and left overnight. The next day, secondary anti-rabbit antibodies were added, and samples were incubated at room temperature for 1 hour. After incubation with ECL luminescent solution (Millipore), images were acquired using Tanon-5200 (Shanghai, China). ImageJ software was used to analyze the grayscale values of the strips, and β-actin was used as an internal loading control. Further information on the primary antibodies used in this study is shown in Table 2.

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).

Total proteins were extracted from EPCs using radioimmunoprecipitation assay lysis buffer (Bioswamp) supplemented with protease and phosphatase inhibitors. The proteins were quantified using a bicinchoninic acid assay kit (Bioswamp). The obtained proteins (20 μL) were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Millipore). The membranes were blocked with 5% skim milk for 2 h at room temperature and incubated overnight at 4°C with the following primary antibodies: PI3K (Abcam, 1:1000), Akt (Bioswamp, 1:1000), p-Akt (Bioswamp, 1:1000), glycogen synthase kinase 3β (GSK3β, Abcam, 1:5000), p-GSK3β (Abcam, 1:1000), extracellular signal-regulated kinase 1/2 (ERK1/2, Abcam, 1:1000), p-ERK1/2 (Abcam, 1:1000); caspase 3 (Bioswamp, 1:1000), angiopoietin (Ang)1 (Abcam, 1:500), Ang 2 (Abcam, 1:5000), and glyceraldehyde 3-phosphate dehydrogenase [GAPDH] (CST, 1:1000). After washing, the membranes were incubated with a goat anti-rabbit IgG secondary antibody (Bioswamp, 1:20000) at room temperature for 1 h. Immunoreactivity was visualized by colorimetric reaction using enhanced chemiluminescence substrate buffer (Millipore) using an automatic chemiluminescence analyzer (Tanon-5200, Shanghai, China). The band gray values were measured by TANON GIS software.