Western Blotting and Immunostaining of Neonatal Rat Cardiomyocytes

Western blotting was performed from neonatal rat cardiomyocytes using standard procedures. Antibodies used were METTL3 (Bethyl Laboratories), MAP3K6 (Novus Biologicals), MAP4K5 (Thermo Scientific Pierce), MAPK14 (Cell Signaling Technology) and GAPDH (Fitzgerald Industries). Masson’s trichrome staining was performed from histological sections generated from paraffin-embedded hearts. Immunostaining was performed using α-Actinin antibodies (Sigma Aldrich) and cell area was quantified using CellProfiler following published methods.^{24 (link)} Detection of the cell membrane was performed using FITC-conjugated Wheat Germ Agglutinin (Sigma Aldrich). Cross-sectional area was measured using ImageJ. RNA was extracted from neonatal rat cardiomyocytes or mouse hearts using Trizol and reverse transcription was performed using the High Capacity cDNA Reverse Transcription kit (Applied Biosystems). Selected genes and m⁶A peaks were analyzed by real-time PCR using SYBR green (Applied Biosystems). Quantified mRNA expression was normalized to Rpl7 (Ribosomal Protein L7), and expressed relative to controls.