Anti sirt3

Proteins were extracted with radioimmunoprecipitation assay (RIPA) buffer (150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 50 mM Tris-HCl, 2 mM ethylenediamine tetraacetic acid, 1 mM phenylmethylsulfonylfluoride, 1 mM dithiothreitol, 10 mM Na₃VO₄ and 20 mM NaF, pH 7.5). Homogenates were centrifuged at 4 °C for 15 min, and the supernatant was used for western blot. Proteins of 20–50 μg were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, Beyotime, Shanghai, China) and then transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with anti-SIRT3 (1:1000, Santa Cruz Biotechnology Inc., San Diego, CA, USA), anti-ANP, anti-BNP, anti-forkhead-box-protein 3a (FOXO3a), anti-SOD2 (1:1000, Abcam, Cambridge, UK), or anti-β-tubulin (1:3000, Bioworld Technology, St. Louis, MO, USA) primary antibodies overnight at 4 °C. After washing with TBST, the membranes were incubated with a horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology Inc., San Diego, CA, USA) for 2 h at room temperature. Finally, the membrane was exposed to enhanced chemiluminescence substrate (ECL, Thermo Fisher Scientific Inc., Rockford, IL, USA) reagent for determination of protein expression.

Total protein was harvested from cells using cell lysis buffer (50 mM Tris-HCl (pH 8.0), 4 M urea, and 1% Triton X-100) supplemented with protease inhibitors. To determine the concentration of cell lysates, the BCA kit (KeyGEN BioTECH, Cat. No: KGP902) was employed. Subsequently, proteins of varying molecular weights were separated through SDS-PAGE and transferred onto a nitrocellulose membrane. Subsequently, the membrane was blocked with 5% skim milk for 2 h and then incubated with primary antibodies overnight at 4 ℃, followed by secondary antibodies for 1 h. The following antibodies were used: anti-ETS1 (Beyotime, Cat. No: AF6812, 1:1,000), anti-E-Cad (ABclonal, Cat. No: A20798, 1:2,000), anti-Vimentin (Proteintech, Cat. No: 10366-1-AP, 1:2,000), anti-Snail1 (Proteintech, Cat. No: 13099-1-AP, 1:1,000), anti-SIRT3 (Santa Cruz, Cat. No: sc-365,175, 1:200), anti-H3K27cr (PTM BIO, Cat. No: PTM-545RM, 1: 1,000), anti-GAPDH (Proteintech, Cat. No: 60004-1-Ig, 1:10,000), anti-H3 (Proteintech, Cat. No: 17168-1-AP, 1:10,000), anti-rabbit IgG (SAB, Cat. No: L3012, 1:10,000), and anti-mouse IgG (SAB, Cat. No: L3032, 1:10,000). The intensity of bands was scanned and then measured using Image J software.

Protein samples extracted from cardiac fibroblasts or the left ventricular myocardium were separated by 10% or 6% sodium dodecyl sulfate- (SDS-) polyacrylamide gel electrophoresis (PAGE). Then, the proteins in the gel were transferred onto membranes of polyvinylidene fluoride (PVDF) (Millipore, Billerica, MA, USA). After the membranes were blocked with 5% nonfat milk at room temperature for 2 h, the membranes with proteins were incubated overnight with an appropriate primary antibody for anti-SIRT3 (1 : 1000; Santa Cruz Biotechnology, St. Louis, MO, USA), anti-α-SMA, collagen I, collagen III (1 : 1000; Bioss, Beijing, China), anti-DRP1 (1 : 1000; Cell Signaling Technology, USA), anti-β-tubulin (1 : 3000; CMCTAG, Milwaukee, WI, USA), and anti-GAPDH (1 : 7000; Sigma-Aldrich, St. Louis, MO, USA) at 4°C. Next, membranes were incubated with horseradish peroxidase- (HRP-) labeled IgG at room temperature for 2 h. Enhanced chemiluminescence (ECL, Thermo Fisher Scientific Inc., Rockford, IL, USA) was dropped to visualize the protein blots.

Protein samples were separated with sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). After blocking, the primary antibodies were incubated overnight at 37 °C followed by HRP-conjugated secondary antibodies incubation for 2 h. The primary antibodies used were listed as follows: anti-SIRT3 (1:1000, Santa Cruz Biotechnology Inc., San Diego, CA, USA), anti-SOD2 (1:1000, Abcam, Cambridge, UK), and anti-β-tubulin (1:3000, Bioworld Technology, St. Louis, MO, USA). Protein bands were visualized with enhanced chemiluminescence (ECL) (Thermo Fisher Scientific Inc., Rockford, IL, USA). The target protein expression level was normalized to the level of β-tubulin.