Enhanced chemiluminescence method

With density gradient centrifugation, cytosolic and nuclear fractions were separated after H9c2 cells from each group were lysed in RIPA buffer (Beyotime, Shanghai, China). Subsequently, the mixture was centrifuged for 15 min (12,000 r/min, 4°C) and the supernatant was collected, which was considered as the protein sample, before protein concentration was measured using Bicinchoninic Acid (BCA) Protein Assay Kit (Boster, Wuhan, China). After the protein samples were denatured, 20 μg of protein sample in each group was dissolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then the proteins were transferred to polyvinylidene fluoride (PVDF) membrane (Beyotime, Shanghai, China). Following that, the PVDF membrane was blocked with 5% skim milk for 2 h and then incubated with anti-histone H3 (Abcam, ab215728, Cambridge, UK, 1:1000), anti-GAPDH (Abcam, ab8245, Cambridge, UK, 1:1000), anti-STAT3 (Proteintech, 10253-2-AP, Wuhan, China, 1:1000) and anti-p-STAT3 (Abcam, ab76315, Cambridge, UK, 1:500) primary antibodies at 4°C overnight. Next, the membrane was incubated with the secondary antibody (Proteintech, 10253-2-AP, Wuhan, China) at room temperature for 2 h. Finally, the protein bands were detected with the enhanced chemiluminescence method (Beyotime, Shanghai, China).

Total protein was extracted from tissues and cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich, St. Louis, MO, USA). The protein concentration was determined by the BCA assay kit (Beyotime, Haimen, Jiangsu, P.R. China). The same amount of protein (50 μg) was separated using 10% SDS-PAGE gel, and then the protein was transfected to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with fat-free milk and incubated with antibody against GATA6 (1:1,000; ab106066; Abcam, Cambridge, MA, USA) and GAPDH (1:1,000; ab8245; Abcam) at 4°C overnight. Finally, the membrane was incubated with HRP-conjugated rabbit anti-mouse IgG (1:5,000; ab6728; Abcam). The protein was detected with the enhanced chemiluminescence method (Beyotime). Protein signals were quantified using Quantity One Software (Bio-Rad, Hercules, CA, USA).

Proteins from cell lysates (20 µg) were separated by 10% SDS‐polyacrylamide gel electrophoresis and transferred to a polyvinylidene difluoride membrane. After blocking in 5% nonfat milk, protein blots were incubated with specific antibodies followed by incubation with a peroxidase‐conjugated secondary antibody in blocking buffer. The bands were visualized with the enhanced chemiluminescence method according to manufacturer's instructions (Beyotime Institute of Biotechnology).