Horseradish peroxidase hrp labeled secondary antibody

The total proteins were isolated from cardiac fibroblasts that were western blotted using the monoclonal antibodies against EGFR (1 : 1, 000, Abcam, Cambridge, MA, USA), ERK1/2 (1 : 1, 000, Affinity, USA), and p-ERK1/2 (1 : 1, 000, Affinity). The loading control was served by GAPDH (1 : 1, 000, Sigma-Aldrich, USA). Cardiac fibroblasts were cultured at 25°C for 1 h with a horseradish peroxidase (HRP) labeled secondary antibody (1 : 1,000, Proteintech, Chicago, USA). Band density quantifications were investigated using an Odyssey Infrared Imaging System (LI-COR Biosciences, Lincoln, NE, USA).

The proteins from the cultured cells were harvested by using a phenylmethylsulfonyl fluoride (PMSF) lysate buffer. Samples containing 50 μg of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) and then transferred to polyvinylidene fluoride membranes. After the membranes were blocked with 5% non-fat milk for 1 h, they were incubated with the primary antibody at 4 °C overnight: NEIL3 (ab230908; Antibody Cambridge (Abcam)), p-ATR (2853S; Cell Signaling Technology (CST)), p-ATM (5883S; CST), γ-H2AX (9718S; CST), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (2118S; CST). The membranes were then incubated with the horseradish peroxidase (HRP)-labeled secondary antibody (Proteintech, Wuhan, Hubei, China) at 37 °C for 1 h. The protein band signal was detected using the BeyoECLPlus chemiluminescence reagent (Beyotime, Shanghai, China).

Total proteins were extracted by ice-cold-RIPA lysis buffer and protein concentrations were quantified using a BCA kit (Pierce, Rockford, IL, USA). The samples were separated by 8–10% SDS-PAGE gel (Beyotime, China) and then transferred onto PVDF membranes. After blocking with 5% BSA at room temperature for 2 h, the membranes were incubated with primary antibodies overnight at 4 °C. Subsequently, the horseradish peroxidase (HRP)-labeled secondary antibody (Proteintech Group, Inc.) was used to cover the membranes for 50 min at room temperature. The blots were developed using enhanced chemiluminescence. The primary antibodies were as follows: ING5 (Proteintech Group, Inc., Rosemont, IL, USA), PI3K, p-PI3K, AKT, p-AKT, MEK, and p-MEK (Cell Signaling Technology, Danvers, MA, USA), and GAPDH (Proteintech Group, Inc.). GAPDH was used as the loading control.

For western blot (WB) analysis, the purified extracellular vesicles were lysed with radio-immunoprecipitation assay buffer (Santa Cruz Biotechnology, Dallas, TX, USA), and the cleared lysate was collected by centrifugation for protein separation on 12% sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gels. After electrophoresis, the separated proteins were transferred to 0.45 μm polyvinylidene difluoride (PVDF) membranes (Millipore, USA). The membranes were blocked for 1 h with Tris-buffered saline containing Tween 20 (TBST) with 5% non-fat milk. The blots were then incubated with primary antibody at 4°C overnight. The primary antibodies used included mouse monoclonal anti-CD63 (Abcam, Cambridge, UK), anti-ASFV p30 (Prepared by our laboratory), rabbit monoclonal anti-CD9 (Abcam, Cam bridge, UK), anti-APOA1(Abcam, Cam bridge, UK), rabbit polyclonal anti-ASFV p72 (prepared by our laboratory), SERPINC1 (Abcam, Cam bridge, UK). After washing three times with TBST, the membranes were incubated with horseradish peroxidase (HRP)-labeled secondary antibody (Proteintech, Chicago, IL, USA) for 2 h at room temperature. Finally, the proteins were visualized with Clarity enhanced chemiluminescence (ECL) WB substrate (Bio-Rad Laboratories, Hercules, CA, USA).

Protein lysates of mouse brain tissue were prepared in RIPA:PMSF (100:1) supplemented with phosphatase inhibitors. Protein concentrations were measured by the bicinchoninic acid assay (Beyotime, Shanghai, China). Samples were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes (Pall, East Hills, NY, United States). The PVDF membranes were blocked with 5% skim milk and incubated with primary antibodies reactive with KOR (1:1000; Abcam, Cambridge, MA, United States) or beta-actin (1:1000; Beyotime), at 4°C overnight. The blots were then incubated with horseradish peroxidase (HRP)–labeled secondary antibody (Proteintech, Shanghai, China) for 1 h. The membrane was incubated with SuperSignal West Pico Chemiluminescent Substrates (Thermo Fisher Scientific, Waltham, MA, United States) and bands visualized using an automatic multifunction chemiluminescent detection system (Tanon, Shanghai, China). The signals were calculated by densitometry using ImageJ software.