Immobilon classico western hrp substrate

N2a and ScN2a were rinsed in PBS 1X and resuspended in lysis buffer (10 mM Tris HCl, 150 mM NaCl, 0.5% NP-40, 0.5% sodium deoxycholate). Lysates were centrifuged for 5 min at 5900g at 4 °C. Total protein content of N2a and ScN2a cells was quantified using bicinchoninic acid assay (Sigma-Aldrich). Proteins (20 μg) were diluted in loading buffer and samples boiled for 10 min at 100 °C.
Samples were loaded in 12/15% Tris-Glycine SDS-PAGE gels and transferred onto Immobilon P PVDF membranes (Millipore) for 2 h at 4 °C. Membranes were blocked in 5% nonfat milk in TBST and incubated overnight at 4 °C with mouse anti-PrP W226 1:1000 (kindly provided by Prof Robert Kammerer) (43 (link)), mouse anti-tau 7.51 1:500 (44 (link)), and rabbit anti-LC3B 1:1000 (2775, Cell Signaling) antibodies. After three washes in TBS-T, membranes were incubated with goat-anti-mouse IgG horse radish peroxidase (HRP)-conjugated antibody (Dako) for 1 h at room temperature (RT) and developed using Immobilon Classico Western HRP substrate (Millipore). Mouse anti β-actin-HRP antibody 1:10.000 (A3854, Sigma-Aldrich) was incubated for 1 h at RT and used for normalization. Images were acquired using Uvitec Alliance (Cambridge), and Uviband software was used for densitometric analysis.

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).

Protein expression was determined by immunoblotting. Briefly, cell pellets were prepared in radioimmunoprecipitation assay (RIPA) buffer (Sigma) in the presence of a protease inhibitor cocktail (Roche). Total protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. Forty micrograms of protein were electrophoresed on 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon-FL membranes (Millipore). Membranes were blocked in tris buffered saline with Tween-20 (TBS-T; 50 mM Tris/HCl, 150 mM NaCl, pH 7.5 plus 0.2% Tween-20) and 5% non-fat milk for 1 h at room temperature. Blots were probed with the following primary antibodies: rabbit anti-OGG1 (ab124741, Abcam) at 1/2500 dilution, and mouse anti-β-actin (A5441; Sigma) at 1/10,000 dilution in TBS-T containing 5% non-fat milk. Anti-mouse and anti-rabbit IgG-HRP (immunoglobulin G horseradish peroxidase; Dako) were used as the secondary antibodies, and the immunoblots were developed using Immobilon Classico Western HRP substrate (Millipore). Each immunoblot was performed in triplicate. Images were analyzed using ImageJ software (NIH Image), and OGG1 protein level was normalized to actin levels. The full-length blots are presented in Supplementary Figure S2.

Approximately 1 × 10⁶ cells were plated 24 h before collection. For chloroquine treatment, cellular pellets were lysed with RIPA buffer (5 M NaClO, 25 M Na₂HPO₄, 0.5 M NaH₂PO₀, 0.5 M EDTA pH 8, 10% NaDOC, 10% Triton X-100, and 10% SDS), and 1:100 protease inhibitors were added before use. The lysed cells were maintained on ice for 20 min and then sonicated for 10 s continuously at an output of 2. After centrifugation for 10 min at 4 °C and 13,000 rpm, the supernatant was collected, quantified and stored at −80 °C. The protein concentration was measured using the bicinchoninic acid assay method. Equal amounts of proteins were separated by 12% Tris-HCl SDS‒PAGE. The following primary antibodies were used: β-tubulin (T7816, 1:5000), LC3B (L7543, 1:1000), and p62 (P0067, 1:1000). Immobilon® Classico Western HRP substrate (Millipore) was used to reveal proteins. Chemiluminescent detection was performed on an LAS-4000 mini instrument (Fujifilm, Düsseldorf, Germany). The quantifications were performed using ImageJ 1.45 software.

The 0.3-µL samples obtained via SEC ware spotted onto the nitrocellulose membrane. The fluorescence intensity of the CM-Dil dye was measured on an Invitrogen iBright FL1500 Imaging System with an exposure time of 0.016 s. The values were digitized using the ImageJ program. To assay the protein content of SEC-separated plasma fractions, the membrane with spotted samples was dried, blocked by soaking in 5% BSA in TBS-T in RT for 30 min, and incubated at 4 °C overnight with primary antibodies against CD9 (HBM-CD9-100, HansaBioMed, Tallin, Estonia), CD63 (ab68418, Abcam, Cambridge, UK), HSP70 (kindly provided by Dr. A. Zhahov, RU patent 2 722 398), HSA (4T24, HyTest, Turku, Finland), Reg IV (ABP56724, Abbkine, Wuhan, China), and DHRS11 (ABP56569, Abbkine, Wuhan, China). Primary antibodies were dissolved to 1 mkg/mL in 0.1% BSA in TBS-T. Then, the membrane was washed three times with TBS-T (3 × 5 min), incubated at 4 °C for 30 min with secondary HRP-conjugated Fc fragment-free antibodies (ab6823, ab7171, AbCam, Cambridge, UK), diluted at a ratio of 1:25,000 in 0.1% BSA in TBS-T, and washed three more times with TBS-T (15 min × 2 times), then once with TBS (5 min). The membrane was soaked in Immobilon Classico Western HRP substrate (WBLUC0020, Millipore, Burlington, MA, USA) in the dark for 5 min. The images were obtained using the Invitrogen iBright FL1500 Imaging System.