Ab2254

Hearts were embedded in paraffin and cut in 5 μm sections, deparaffinized, rehydrated and antigen retrieval. Sections were permeabilized with 0.5% Triton X-100/PBS and then blocked with 5% goat serum (Jackson ImmunoResearch Laboratories, USA) for 1 hr at room temperature, and incubated with primary antibodies overnight at 4℃. After washing with PBS, sections were incubated with corresponding secondary antibodies conjugated to fluorescence for 1 hr at room temperature, followed by counterstaining with DAPI (Sigma). Primary antibodies are as follows: anti-Mettl3 (Abcam, ab195352, 1:500), anti-m⁶A (Synaptic Systems, 202111, 1:100), anti-Ki67 (Abcam, ab16667, 1:250), anti-phospho-Histone H3 (pH3) (CST, #53348 S, 1:400), anti-aurora kinase B (AurkB) (Abcam, ab2254, 1:200,) anti-GFP (Proteintech, 50430–2-AP, 1:200), and anti-Cardiac Troponin T (cTnT) (ThermoFisher, MA512960, 1:200). Secondary antibodies used are following: Alexa Fluor 488 goat anti-mouse or anti-rabbit IgG (Jackson ImmunoLabs, 115-545-071 or 111-545-003, 1:200), and Cy3-conjugated Affinipure Goat anti-mouse or anti-rabbit IgG (Proteintech, SA00009-1 or SA00009-2, 1:300). The slides were imaged with fluorescence microscope (Leica Microsystems) or Zeiss LSM 700 laser confocal microscope (Carl Zeiss).

Total protein was extracted with RIPA lysate, and cytoplasmic/nuclear proteins were extracted using the Nuclear and Cytoplasmic Protein Extraction Kit (TransGen Biotech). After determining the concentration by the BCA method, proteins were heated at 99°C for 10 min, subjected to SDS‐PAGE gel electrophoresis, and transferred to the PDVF membrane. Immunoblots were carried out with primary antibodies, including anti‐NFAT5 (Abcam, ab3446), anti‐AURKB (Abcam, ab2254), anti‐Phospho‐Ser/Thr (Abcam, ab17464), anti‐AQP4 (Abcam, ab2254), anti‐GAPDH (ZENBIO, 20030–67E4), anti‐Histone 3 (CST, 4499s).
Co‐IP assay was performed using a Direct Magnetic IP/CO‐IP Kit (Thermo Fisher Scientific, 88 824). Briefly, rat SDH tissue was lysed with IP lysis buffer supplied with protease and phosphatase inhibitors, and then incubated with the primary antibodies: anti‐NFAT5 (Novus Biologicals, NB1203446), or anti‐AURKB (Abcam, ab2254), or anti‐IgG (Proteintech, B900610). Magnetic beads (Thermo Fisher Scientific, 88 824) were used to pull down the corresponding protein complex. After eluted the proteins from the beads, and western blot was used to identify the specific protein.

For Western blot analysis, the total proteins of samples were isolated by RIPA lysis buffer. The protein samples were prepared with a 5 × sample loading buffer and resolved on sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels. Transferred NC membranes were probed with antibodies against Aurkb (Abcam, ab2254, 1:1,000, 0.001 g/μL) and GAPDH (Proteintech, 10494-1-AP, 1:1,000, 0.001 g/μL). Chemiluminescent signals were detected by the LiCor Odyssey Fc instrument.