Protein Extraction and Western Blot Analysis

The proteins extraction was prepared from H9c2 cells [20 (link)]. Then, proteins were lysed in the RIPA lysis buffer (Elabscience, Wuhan, China) on ice. The quantitation of proteins concentration was conducted with the aid of the BCA Protein Assay kit (Beyotime, Shanghai, China) in the light of the guidance of manufacturer. Subsequently, the protein samples were separated by SDS-PAGE gels, followed by the shifts onto polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After washing with PBS and blocking with 5% skimmed milk, primary antibodies (SFRP4, 1:1000, ab154167; IL-1β, 1:1000, ab254360; TNF-α, 1:1000, ab215188; PTPN12, 1:1000, ab289859; PI3K, 1:1000, ab191606; AKT, 1:1000, ab179463; p-PI3K, 1:1000, ab182651; p-AKT, 1:1000, ab192623; GAPDH, 1:1000, ab8245) were put in the incubator and cultivated with these membranes overnight at room temperature, followed by an incubation of secondary antibodies labeled by horseradish peroxidase at indoor temperature for 1 h. The detection of protein bands was implemented through Image J software (National Institutes of Health, Bethesda, MD, USA).