Ythdf3

Protein extraction of left ventricle tissue and the protein concentration was determined by Bradford method (M&C Gene Technology Ltd.). The protein samples were then subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane. The membranes were probed with antibodies of interest. The antibodies used were as follows: METTL3, METTL14, Wilms tumor 1-associated protein (WTAP), and YTH N6-methyladenosine RNA binding protein 1 (YTHDF1), YTHDF3 (all1:1,000; Cell Signal Technology), and β-actin (1:4,000; Abcam).

Total protein extracts were isolated with Radioimmunoprecipitation assay (RIPA) buffer in the presence of protease and phosphatase inhibitor cocktail (Roche). Equal quantities of protein were separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), transferred to nitrocellulose membranes, immunoblotted with the appropriate antibodies, and revealed using Odyssey. The antibodies used in this study were as follows: METTL16 (Cell Signaling Technology, #87,538), PRDM15 (Proteintech, 25,590–1-AP), SETD5 (Abclonal, A7304, RRID:AB_2767845), KMT2D (Proteintech, 27,266–1-AP), NSD2 (Abclonal, A7938, RRID:AB_2772896), FGFR4 (Proteintech, 67,800–1-Ig), YTHDF1 (Proteintech, 17,479–1-AP, RRID:AB_2217473), YTHDF3 (Cell Signaling Technology, #24,206), FLAG (Cell Signaling Technology, #14,793), HA (Cell Signaling Technology, #3724), H3K27ac (Cell Signaling Technology, #8173), p300 (Cell Signaling Technology, #54,062), YY1 (Cell Signaling Technology, #46395S), β-actin (Sigma, A2228), and the anti-rabbit and anti-mouse secondary antibodies (LI-COR, Ride® 800CW).