β actin

Proteins were obtained using radioimmunoprecipitation assay (RIPA) buffer to dissolve the cells. The quantification was tested using bicinchoninic acid Protein Assay Kit (Boster Biological Company, Ltd., Wuhan, China). All protein samples were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The target strip was transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA) via electrophoresis. The PVDF membrane was blocked in 5% nonfat milk. The protein bands were incubated with specific primary antibodies as follows: HNRNPH1 (1:2,000) (ab 154894, Abcam, CA, USA), PTPN6 (1:1,000) (ab 32559, Abcam, CA, USA), AKT (1:1,000) (ab 38449, Abcam, CA, USA), p-AKT (1:1,000) (ab 8805, Abcam, CA, USA), and β-ACTIN (1:8,000; Abways Technology, New York, NY, USA; AB0035). Moreover, the PVDF membrane was incubated in a goat-anti-rabbit secondary antibody (1:10,000, Boster Biological Company, Ltd., Wuhan, China) overnight. Images of protein quantification were captured by BioSpectrum Imaging System (UVP, LLC, Upland, CA, USA).

Total proteins in spleens or aortas were extracted via High Efficiency RIPA Lysis Buffer (KeyGEN BioTECH), quantified by BCA protein concentration assay kit (Beyotime Biotechnology), and denatured in boiling water with SDS-PAGE protein loading buffer (Beyotime Biotechnology). 30 μg of proteins per sample was loaded in 10% SDS-PAGE gel and transferred onto polyvinylidene fluoride membranes (Merck Millipore). Skim milk in TBST (TBS containing 0.1% Tween 20) was used as blocking agent before antibody incubation. Polyvinylidene fluoride (PVDF) membranes were incubated with rabbit antibodies against p-STAT3-Y705 (Abcam), p-STAT5-Y694 (Cell Signaling Technology), SOCS3 (Abcam), FOXP3 (Abcam), β-ACTIN (Abways), and Goat anti-rabbit IgG with HRP (Biosharp), respectively. The imaging was performed in Gel Doc XR Biorad (Bio-Rad) using Chemiluminescent HRP Substrate (Merck Millipore). Blot data were analyzed with Image Lab 4.0 (Bio-Rad). Gray values of blot areas are measured, and the relative expression amount of the protein samples is calculated by the method of the target protein gray value/internal reference β-ACTIN gray value.

BHK-21 cells (1 × 10⁵ cells per well) were seeded into six well culture plates, incubated overnight and transfected with 50 nM of the miR-3470b mimics or 50 nM of the miR-3470b inhibitor or their respective non-targeting negative control oligonucleotides using Attractene Transfection Reagent (Qiagen, Germany). Meanwhile, BHK-21 cells was infected with BEFV at a multiplicity of infection (MOI) of 0.1 were harvested at 36 h post-transfection and subjected to analyze MAVS protein levels with rabbit anti-MAVS (CST, Proteintech Group, Chicago, IL, USA) and β-actin (Abways, China) antibodies for western blotting analysis. The blots were detected by ECL reagent (Thermo Scientific, USA) and scanned by the enhanced chemiluminesence detection system (Amersham, USA). Image J software was used for quantifications of protein blot intensities by gray value analysis (National Institutes of Health, Bethesda, MD, USA).

Cardiomyocytes or HuMSC-EVs were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer prior to whole-protein purification. The protein concentration was measured by a bicinchoninic acid assay (BCA) kit (Beyotime Institute of Biotechnology, Haimen, China). Equal amounts of protein underwent 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were incubated with the primary antibody overnight at 4°C and then incubated with a horseradish peroxidase-conjugated goat anti-rabbit IgG secondary antibody (1:2000, Ca#BE0101, EASYBIO, Beijing, China) for 1 h at room temperature. Specific protein bands were observed with an ECL Plus chemiluminescence kit (EMD Millipore) and quantitatively analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). Experiments were carried out in triplicate. The primary antibodies used in this study were as follows: Bax (1:5000, Ca #ab32503, Abcam, Cambridge, MA, USA), Bcl-2 (1:2000, Ca #ab196495, Abcam); cleaved-caspase 3 (1:1000, Ca#184787, Abcam), and β-actin (1:5000, Ca#0035, Abways, Shanghai, China).