Chemiluminescence detection system

Western blot was performed as described previously [35 (link)]. GMECs were lysed with ice-cold RIPA buffer (Solarbio, Beijing, China) containing protease and phosphatase inhibitor cocktail (Solarbio, Beijing, China). The protein concentrations were examined with a BCA kit (Epizyme, Shanghai, China). The quantified protein from each group was separated by 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, USA). Following blocking with 5% skimmed milk in Tris-Buffered Saline and Tween 20 (TBST) buffer for 2 h at room temperature, the membranes were incubated with primary antibodies at 4 ℃ overnight. After washing five times with TBST for 5 min each time, the membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. The blots were washed three times with TBST and determined with a chemiluminescence detection system (Bio-Rad Laboratories, Hercules, CA, USA). The levels of target protein bands were quantified with Image J software. The primary antibodies used for Western blot are listed in Table 2.

The cells were taken from the incubator to remove the culture medium and washed twice with precooled PBS. Proteins were harvested using whole cell lysis assay (KeyGEN Biotech, China) according to the manufacturer's instructions. After valuing the concentration and equaled the quantity of loading samples, the proteins of different groups were separated by SDS-PAGE and subsequently transferred onto PVDF membranes (Millipore, MA, USA). Afterwards, the membranes were blocked with 5% nonfat milk in TBST at room temperature for 1 h, followed by incubation with antibodies against targeting protein overnight at 4°C. The primary antibodies including anti-CCR5, anti-TRAF6, anti-NF-kB, and anti-PI3K were purchased from Abcam (Cambridge Science Park, UK), and used as the following concentration: anti-CCR5: 1 : 1000; anti-TRAF6:1 : 2000; anti-NF-kB:1 : 500; anti-PI3K:1 : 250; and anti-β-Actin:1 : 5000. Following being washed with TBST buffer, the membrane was stripped with appropriate HRP-conjugated secondary antibodies (Abcam, CA, USA) for 1 h at room temperature, followed by visualized using the enhanced Western Bright ECL reagents (Cell Signaling Technology, USA). Eventually, bands were imaged and analyzed by a chemiluminescence detection system (Bio-Rad, USA). The experiments were repeated at least three times to ensure reproducibility.

EC cells were harvested and lysed in RIPA lysis buffer (Servicebio). After extraction, the total protein was measured by the Bicinchoninic Acid (BCA) protein assay kit (Beyotime), mixed with 5 × loading buffer, and then boiled in 100°C water for about 10 min. Subsequently, the well‐prepared samples were loaded into SDS‐PAGE with constant voltage and transferred to Polyvinylidene fluoride (PVDF) membranes with a constant current. Following blocking with 5% skim milk (Servicebio) for about 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4°C. The following primary antibodies were used in this research: Rabbit anti‐human RAC3 antibody (1:1000 dilution; Abcam) and rabbit anti‐human β‐actin antibody (1:1000 dilution; Abcam). Then, membranes were incubated with the appropriate Horseradish Peroxidase (HRP)‐conjugated secondary antibodies (1:5000 dilution; Abcam) for 1 h at room temperature. Finally, protein bands were detected using the chemiluminescence detection system (Bio‐Rad).

Total proteins of fresh liver tissues in each group were extracted using a whole cell lysis assay kit (KeyGEN BioTECH, Nanjing, China) and the protein concentration was determined using the bicinchoninic acid (BCA) protein quantitation assay kit (KeyGEN BioTECH, Nanjing, China). Equal amounts of lysates were separated by 12% SDS-PAGE gel electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. After blocking in 5% (w/v) non-fat dry milk in PBST for 1 h, the membranes were incubated with primary antibodies for detecting cytochrome C, Bcl-2, Bax, caspase-3, caspase-8, caspase-9, TLR4, TRIF, TAF6, MyD88, p65, NF-κB, and Iκ-Bα (Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. Then, the membranes were washed with TBST and incubated with HRP-conjugated secondary antibodies at room temperature for 1 h. Protein bands were visualized with ECL and determined using a chemiluminescence detection system equipped with imaging software (Bio-Rad Laboratories, Philadelphia, PA, USA).

Cytoplasmic and nuclear extracts were prepared using a Nuclear Extract kit (Active Motif, Carlsbad, CA, USA) according to the manufacturer's recommendations. Equivalent amounts of protein were separated by 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore Co., Billerica, MA, USA). After being blocked in 5% non-fat milk in TBST [10 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.1% Tween 20] for 2 h at room temperature, the membranes were incubated with the appropriate primary antibodies at 4°C overnight. The immunoblots were then incubated with a secondary antibody conjugated with horseradish peroxidase for 1 h at room temperature. The membranes were developed using an electrochemiluminescence (ECL) kit (Thermo Scientific/Pierce, Rockford, IL, USA) according to the manufacturer's protocol. The signals were detected by a chemiluminescence detection system (Bio-Rad Laboratories, Hercules, CA, USA). The density of the immunoreactive bands was analyzed using ImageJ 1.41 (National Institutes of Health, Bethesda, Maryland, USA).

Cells were cultured in 96-well plates and lysed in cold RIPA buffer (MDL biotech, Beijing, China) with protease inhibitors. Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene fluoride membranes (Millipore, USA). Membranes were blocked for 1 h at room temperature using 5% fetal bovine serum in tris-buffered saline tween (TBST). Following incubation, membranes were probed with FGF-23, FGFR-1, Klotho, Col-X and actin primary antibodies (MDL biotech, Beijing, China) at 4 °C overnight. After washing 3 times with TBST, membranes were incubated with secondary antibodies for 1 h at room temperature. Finally, membranes were washed 3 times with TBST, and data were captured with a chemiluminescence detection system (Bio-rad, USA).

Cells and purified EVs lysis were performed as follows: whole cell lysate was lysed in RIPA buffer containing protease and phosphatase inhibitors (Sigma Aldrich Corporation), then clarified by centrifugation at 9500 g for 10 min at 4 °C, while isolated EVs were prepared in a reducing (for LGALS3BP, Alix and β‐actin immunoblotting) or non‐reducing (for CD9 and CD63 immunoblotting, without 2‐mercaptoethanol) sample buffer (50 mm Tris–HCl pH 6.8, 5% glycerol, 2% SDS, 1.5% 2‐mercaptoethanol with bromophenol blue) and heated at 95 °C for 10 min. Cellular protein amount of cellular and EVs lysates was determined using the Bradford reagent (Bio‐Rad), and equal amounts of proteins were separated on 10% or 12% SDS/PAGE and transferred to nitrocellulose membrane (Merck Millipore, Germany). The membranes were blocked in 5% skimmed milk in Tris‐buffered saline buffer with 0.1% Tween‐20 for 1 h at room temperature, followed by incubation with primary antibodies overnight at 4 °C. Subsequently, the membranes were incubated with HRP‐conjugated secondary antibodies, detected by enhanced chemiluminescence solution (Pierce, Thermo Fisher Scientific, Cambridge, MA, USA) using a chemiluminescence detection system (Bio‐Rad).