Anti bnp

The proteins extracted from the myocardial tissue were subjected to SDS-PAGE and transferred onto the PVDF membranes (Millipore, Billerica, MA, USA). After blocking with TBST buffer (Tris-HCl 10 mmol·L⁻¹, NaCl 120 mmol·L⁻¹, Tween-20 0.1%; pH 7.4) containing 5% skimmed milk (v/v) at room temperature for 2 h, the membranes were incubated with primary antibodies at 4 °C overnight, including anti-ANP (1:1000, Abcam, Cambridge, UK), anti-BNP (1:1000, Abcam, Cambridge, UK), anti-ox-CaMKII (1:1000, Millipore, Kenilworth, NJ, USA), anti-CaMKII (1:1000, Abcam, Cambridge, UK), anti-caspase-3, anti-cleaved caspase-3, anti-MLKL, anti-p-MLKL,anti-RIPK3 and anti-RIPK1 (1:1000, Cell Signaling Technology, Danvers, MA, USA); anti-p-CaMKII (1:1000, Thermo Fisher Scientific, Rockford, IL, USA), anti-SRSF1(ASF/SF2), anti-SRSF2(SC-35), anti-GAPDH (1:5000, Sigma-Aldrich, St. Louis, MO, USA), and anti-β-tubulin. Subsequently, the blots were incubated with horseradish peroxidase (HRP-)-conjugated secondary antibody at room temperature for 1.5 h. The immunoreactive bands were visualized using enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, Rockford, IL, USA). GAPDH or β-tubulin was used as the loading control.

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 prepared in ice-cold RIPA buffer (150 mM Tris-HCl, pH = 7.6, 150 mM NaCl, 0.5% Triton X-100, 0.1% SDS, 1 mM phenylmethanesulfonyl fluoride, 1 mM Na₃VO₄) containing a cocktail of protease inhibitors (Thermo Fisher Scientific, Waltham, MA, USA) and then quantified using a BCA Protein assay kit (Thermo Fisher Scientific). Protein extracts were resolved on a 10% SDS-polyacrylamide gel and electrophoretically transferred onto a polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA, USA). The following primary antibodies were used: anti-ANP (Abcam, Cambridge, UK; dilution 1:1,000), anti-BNP (Abcam; dilution 1:500), anti-β-MHC (Abcam; dilution 1:1,000), anti-MEF2C (Abcam; dilution 1:1,000) and anti-β-actin (Abcam; dilution 1:3,000). The horseradish peroxidase-conjugated anti-rabbit (Abcam; dilution 1:5,000) or anti-mouse (Abcam; dilution 1:5,000) IgG was used as a secondary antibody. Protein bands were detected using the enhanced chemiluminescence (ECL) detection kit (Immobilon Western Chemiluminescent HRP Substrate, Millipore) and analyzed using ImageJ software (National Institutes of Health, Bethesda, MD, USA).