Anti p camkii

Cardiomyocyte protein was extracted by NP-40 Lysis Buffer (Beyotime, Shanghai, China) and PMSF (Beyotime, Shanghai, China). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membrane (Millipore, Burlington, MA, USA). After being blocked by 5% skim milk for 2 h, the PVDF membrane was incubated with the primary antibody at 4 °C overnight. The primary antibody solution included: anti-RIPK3 (1:1000, Novusbio, Littleton, CO, USA), anti-caspase 3, anti-Cleaved-caspase3 (1:1000, Cell Signaling Technology, Danvers, MA, USA), anti-CaMKII (1:1000, Abcam, Cambridge, UK), anti-ox-CaMKII (1:1000, Millipore, NJ, USA), anti-p-CaMKII (1:1000, Thermo Fisher Scientific, Rockford, IL, USA), anti-GAPDH (1: 5000, CONSON, Shanghai, China). Then, the PVDF membrane was incubated with horseradish peroxidase (HRP)-conjugated IgG (ZSGB-BIO, Beijing, China) for 2 h. The enhanced chemiluminescence solution (ECL, Thermo Fisher Scientific Inc., Rockford, IL, USA) was dropped on the PVDF membrane to observe the protein band.

For western blot analysis, solei were homogenized in reducing sample buffer (80 mM Tris-HCl, pH6.8, 2% sodium dodecyl sulfate, 10% glycerol and 0.1 M dithiothreitol) supplemented with Halt protease and phosphatase inhibitor cocktail (ThermoFisher Scientific). The following antibodies were used for western blot analysis: anti-HSF-1 (Santa Cruz Biotechnology), anti-hsp70 (StressMarq), anti-hsp90 (BD Transduction Laboratories), anti-P-CaMKII (ThermoFisher Scientific), anti-CaMKIIβ (Invitrogen). The quantitative analysis was performed using ImageJ software.

Cells were lysed in ice-cold lysis buffer (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10 mM EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate) supplemented with protease and phosphatase inhibitors. Homogenates were maintained in ice for 30 min and centrifuged at 15 000 × g for 10 min at 4 °C, and the supernatant was recovered. Protein concentration was determined by BCA protein assay kit (Life, New York, NY, USA). Proteins were resolved in SDS-PAGE (10% polyacrylamide), transferred to PVDF membrane, and incubated with primary antibodies. The reactions were followed by incubation with peroxidase labeled secondary antibodies (Life). Primary antibodies used were: anti-RIPK1 (1:800, Abcam), anti-RIPK3 (1:800; Abcam), anti-MLKL (1:1000, Abcam), anti-β-actin (1:5000, Abcam), anti-Na+-K+-ATPase (1:400; Santa Cruz, Santa Cruz, CA, USA), anti-CaMKIIδ (1:800, GeneTex), anti-p-CaMKII (1:800, Thermo), and anti-ox-CaMKII (1:600; Millipore, Bedford, MA, USA). CaMKII activation was assessed by measuring phosphorylation and oxidation levels.

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.