Anti anp

Nucleoproteins were extracted using a nuclear extraction kit (Merck Millipore), separated, subjected to SDS‐PAGE and then transferred to polyvinylidene difluoride membranes using a Bio‐Rad semidry electrotransfer apparatus. The nitrocellulose membranes were blocked with 5% non‐fat milk in Tris‐buffered saline and incubated with monoclonal antibodies (anti‐G9α, anti‐H3K9me3, anti‐MEF2C, anti‐Cx43, anti‐ANP, anti‐β‐MHC and anti‐GAPDH [Abcam]) diluted in Tris‐buffered saline. Protein bands on immunoblots were visualized by enhanced chemiluminescence. After scanning, bands were quantified using Quantity One software version 4.4 (Bio‐Rad).

The proteins extracted from the myocardial tissue were subjected to SDS-PAGE and transferred onto the PVDF membranes (Millipore, Billerica, MA, United States). 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 (1:1,000, Abcam, Cambridge, United Kingdom), anti-BNP (1:1,000, Abcam, Cambridge, United Kingdom), anti-ox-CaMKII (1: 1,000, Millipore, Kenilworth, NJ, United States), anti-CaMKII (1: 1,000, Abcam, Cambridge, United Kingdom), anti-caspase 3, anti-cleaved caspase 3, anti-MLKL, anti-p-MLKL, and anti-RIPK1 (1: 1,000, Cell Signaling Technology, Danvers, MA, United States); anti-p-CaMKII (1: 1,000, Thermo Fisher Scientific, Rockford, IL, United States), anti-GAPDH (1: 5,000, Sigma-Aldrich, St. Louis, MO, United States), 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 by enhanced chemiluminescence (ECL) (Thermo Fisher Scientific, Rockford, IL, United States). GAPDH or β-tubulin was used as the loading control.

Total protein was extracted from tissues or cells using RIPA lysis buffer containing protease inhibitor cocktail (Thermo Fisher Scientific 78,429, Waltham, MA, USA). Lysed cells were centrifuged at 14,000 rpm for 10 mins and the protein concertation in the supernatant was quantified by a BCA Protein assay kit (Beyotime, Shanghai, China). A total of 20 ug protein was used for SDS-PAGE electrophoresis and transferred onto the PVDF membrane (BioRad, Irvine, CA, USA). After blocking with 5% skimmed milk for 1 hour, the membrane was then incubated with primary antibodies: anti-NOVA1 (ab183024, rabbit, 1:1000), anti-ANP (PA5-29,559, rabbit, 1:2000), anti-β-MHC (ab170867, rabbit, 1:1000), anti-BNP (ab19645, rabbit, 1:1000) and anti-GAPDH (ab8245, mouse, 1:2000) antibodies (Abcam, Cambridge, MA, USA) overnight 4°C. After that, the membranes were washed with TBST buffer and further incubated at room temperature with Goat Anti-Rabbit IgG H&L (HRP) (ab6721, 1:2500), or Goat Anti-Mouse IgG H&L (HRP) (ab205719, 1:2500) (Abcam, Cambridge, MA, USA) for 90 minutes. The specific protein bands were developed using an enhanced chemiluminescence kit (Santa Cruz, TX, USA, sc-2048) and photographed on a gel imager system (Bio-Rad, Hercules, CA, United States) [35 (link)]. GADPH served as the loading control.