Anti hdac6

Protein samples were harvested from H9c2 cardiomyocytes after H/R and the myocardium at 24 hours after resuscitation. The separation of cytosolic and nuclear fractions was performed by centrifugation of samples at 16 000g. Subsequently, the samples were separated by SDS‐PAGE, then transferred to a polyvinylidene fluoride membrane, and finally blocked with 5% nonfat milk. After that, the membranes were incubated with primary anti‐NLRP3 (1:1000, Proteintech, Rosemount, IL), anticleaved caspase‐1 (1:1000, Cell Signaling Technology Inc., Danvers, MA), anti–gasdermin D (1:1000, Proteintech), anti‐HDAC6 (1:1000, Proteintech), anti‐GAPDH (1:5000, BBI Life Science Corporation, Shanghai, China), antiacetylated α‐tubulin (1:1000, Proteintech), anti–α‐tubulin (1:1000, Proteintech), anti‐TFEB (1:1000, Proteintech), antiacetylated lysine (1:1000, Cell Signaling Technology Inc.), antihistone H3 (1:1000, Abcam) at 4 °C for 24 hours, then rinsed with TBST solution, and finally incubated with the secondary antibody (1:5000, BBI Life Science Corporation) at room temperature for 1 hour. The protein was visualized with enhanced chemiluminescence substrates and analyzed by Image J software (National Institutes of Health, Bethesda, MD).