Nitrocellulose membrane

HepG2 with a density of 1 × 10⁶ cells/mL was counted and 2 mL/well was added into the six-well plate. After being cultured for 24 h, Na[Os^VI(N)(tpm)₂] at the indicated concentrations was added and reacted for 24 h. Cells were scraped with a scraper, collected, and washed twice with cold PBS. Cells were lysed with a cell lysis buffer, of which the main active component is 1% Triton X-100, with protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Protein quantifications were measured using the BCA Protein Assay Kit (Beyotime) by Infinite M200 (Männedorf, Swiss, Tecan). Equal amounts of cellular proteins were mixed with SDS-PAGE Sample Loading Buffer, 5X (Beyotime), boiled at 95 °C for 5 min, and run on 12–15% separation gel. Protein was transferred to the nitrocellulose membrane (BOSTER). The membrane was blocked with 5% nonfat milk in TBST (1X, 0.1% Tween-20) for 40 min at RT, washed with TBST for 5 s, and then probed with primary antibody at 4 °C overnight followed by secondary antibody. Next, the ECL Plus detection kit (Beyotime) was added and the membrane was visualized using High ChemiDoc XRS (Bio-Rad ChemiDoc XRS+, Hercules, CA, USA).

Intestinal mucosa samples were lysed using RIPA buffer (Roche, Shanghai, China). The concentrations of protein in samples were measured by the bicinchoninic acid (BCA) protein assay kit (Beyotime, Nantong, China). The total protein samples were separated through a 10% SDS polyacrylamide gel and then transferred to a nitrocellulose membrane (Boster, Wuhan, China). The membrane was incubated in 1:10,000 monoclonal mouse anti-beta actin (Bioworld, USA), and 1:1,000 rabbit polyclonal CLDN3 antibodies (Abcam, Shanghai, China) at 4 °C overnight. Then membrane was incubated in 1:10,000 diluted horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (Bioworld, USA) or 1:10,000 diluted HRP-conjugated anti-mouse antibody (Bioworld, USA) for 1 h at room temperature. Tanon™ High-sig ECL Western Blotting Substrate (Tanon, Shanghai, China) was applied to the membrane for 5 min after secondary antibody incubation. The chemiluminescent signals were visualized by the Versa Doc™ imaging system. Signal intensity was quantified using Quantity One software (Bio-Rad, USA). Protein expression levels were normalized with β-actin expression level.

About 100 mg of certain tissues were pooled equally from three 160-day-old pigs for membrane protein extraction using a Membrane and Cytosol Protein Extraction Kit (Beyotime, Nantong), according to the manufacturer's instructions, and protein concentration was determined using an Enhanced BCA Protein Assay Kit (Beyotime, Nantong). The extracted membrane protein was denatured with 5×SDS loading buffer at 95°C for 5 min and stored at −80°C until analysis. 40 µg denatured proteins were separated through a 12% SDS polyacrylamide gel and then transferred to a nitrocellulose membrane (Boster, Wuhan). After incubating in 5% nonfat dried milk for 2 h, the membrane was incubated with 1∶10000 HRP-conjugated monoclonal mouse anti-beta actin (Kangcheng, Shanghai) and 1∶200 polyclonal GPR41 and GPR43 antibodies (Santa Cruz Biotechnology, Texas) at 4°C overnight. The membrane was washed three times in TBST, and then incubated with 1∶10000 diluted horseradish peroxidase-conjugated anti-rabbit antibodies (Sunshinebio, Nanjing) for 1 h at room temperature. The membrane was again washed three times in TBST, after which it was incubated in Pierce Western blotting substrate (Pierce Biotechnology, IL) for 1 min. The chemiluminescent signals were visualized by Fujifilm LAS-4000 (Fujifilm, Tokyo).

Proteins were extracted from cells (RIPA, Beyotime Biotechnology) and tumors (One Step Animal Tissue Active Protein Extraction Kit, Sangon Biotech), and quantification of protein lysates was performed by a bicinchoninic acid assay kit (Boster). After that, the proteins were boiled for 5 min at 100°C in a loading buffer (20 μg) and separated in an SDS-PAGE gel at 80 V for 30 min and then at 120 V for 1 h. After that, the proteins were electrotransferred into a nitrocellulose membrane (Boster) at 300 A for 1.5 h and blocked for 1 h with Tris-buffered saline containing 0.1% Tween 20 (TBST) and 5% fat-free milk. After that, the membranes were incubated with corresponding primary antibodies overnight at 4°C and secondary antibodies for 2 h at room temperature after washing. The membranes were measured by an image analysis system (Image-Pro Plus 6.0, Media Cybernetics) after incubating with a high-signal electrochemiluminescence kit (Fdbio Science).

Western immunoblotting was performed to identify EVs components that were targeted by serum antibodies. The protein marker used was of wide range MW; 3–198 (kDa). Mice sera obtained at the 84^th day from mice of infected control group I and experimental group III (infected, vaccinated) were used as the source of primary antibody. Electrotransfer of proteins from polyacrylamide gels to nitrocellulose membrane (Boster, USA) was done after electrophoresis at 60 volts. Fifteen μl of sample containing EVs was used and 6 μl of marker was loaded. Membrane was blocked using 5% of skimmed milk for one hour. Membrane was probed with serum (1:300 dilution in Tris-buffered saline with 0.5% V/V Tween 20 (TBST)). Membrane was incubated overnight in 4°C on a shaker. Bound antibody was detected by polyclonal goat anti mouse Ig/HRP (Dako Co., Denmark) in 1:1000 dilution in TBST. It was added in 5% skimmed milk in TBST and incubated for 1 hour. Membranes were washed in TBST between each of these steps. Enhanced Chemiluminescence mix was prepared and the membrane was incubated for 1–2 minutes. Results were visualized in a dark room. Gel documentation system (Geldoc-it, UVP, England), was applied for data analysis and more accurate determination of the band molecular weight using Total lab analysis software, (Ver.1.0.1).

Total RNA was extracted using TRIzol and subjected to mRNA separation using a GenElute mRNA Miniprep Kit (Sigma). Global m6A levels in polyadenylated RNAs were measured using an EpiQuik m6A RNA Methylation Quantification Kit (Epigentek) following the manufacturer’s instructions. Briefly, an equal amount of polyadenylated RNAs (200 ng) were coated on the assay wells. Antibody capture solution and antibody detection solution were added sequentially to the assay wells. The m6A levels were measured and calculated based on a standard curve. The m6A dot blotting was conducted as previously described (52 (link)). Polyadenylated RNA samples were loaded into a nitrocellulose membrane (Boster-Bio) fixed with 96-wells Bio-Dot Module (Bio-Rad) and cross-linked using a UV crosslinker. After blocking and incubating with anti-m6A antibodies (1:500; Synaptic Systems), the membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies and then detected using the ECL detection system (Bio-Rad).