Fig. 6

YTHDF2 recognizes the m6A site on Myh7 mRNA to promote its degradation. A Western blotting analysis of MYH7 expression in the primary cardiomyocytes transfected with si-nc or si-YTHDF2 for 24 h, then treated with 50 μg/ml CHX for the indicated times. B RT-PCR analysis of Myh7 mRNA expression in the primary cardiomyocytes transfected with si-NC or si-YTHDF2 for 24 h, then treated with ActD treatment (10 μg/ml) for the indicated times. C Amount of Myh7 mRNA in various polysome fractions was analyzed by RT-PCR. D RIP analysis of the m6A modification on the indicated regions of Myh7 mRNA (5’UTR, 5’Untranslated Region; 3’UTR, 3’Untranslated Region; CDS, Coding sequences). E Schematic diagram of the potential m6A modification sites on Myh7 mRNA. F RIP analysis of the m6A modification on the wild type (WT) or mutant (Mut) Myh7 mRNA CDS-6 region, which was cloned into pGL3-luciferase reporter plasmid. G Cells were co-transfected with pCMV3-C-FLAG-YTHDF2 (YTHDF2), pCMV3-C-FLAG-YTHDF2 mutant (YTH del), or pCMV3-C-FLAG vector (Flag), and pGL3-luciferase-wild type (WT) Myh7 mRNA CDS-6 region, pGL3-luciferase-mutant (Mut) Myh7 mRNA CDS-6 region, or pGL3-luciferase vector (Vector). RIPs were performed to detect the association between YTHDF2 or YTH-del and WT or Mut Myh7 mRNA CDS-6 region. H RNA pulldown analysis of the interaction between biotinylated-Myh7 mRNA CDS-6 (WT) or mutant Myh7 mRNA CDS-6 (Mut) and pCMV3-C-FLAG-YTHDF2 (DF2) or pCMV3-C-FLAG-YTHDF2 mutant (del). ^*P < 0.05