Western blotting analysis

Western blotting analysis was conducted to detect relative protein levels for METTL3, METTL14, FTO, ALKBH5 and YTHDF2 which are associated with RNA methylation. First, all proteins samples were homogenized on ice with RIPA Lysis Buffer (Beyotime, Shanghai, China), which contains 150 mM NaCl, 50 mM Tris-HCl (pH = 7.4), 1% sodium deoxycholate, 1% Triton X-100, 0.1% SDS and some specific protease inhibitors. A BCA Protein Assay Kit (Beyotime, Shanghai, China) was used to detected total protein concentrations of each sample. Subsequently, a total of 30 μg of protein from each tissue was separated by electrophoresis on SDS-PAGE gels. After transferring to nitrocellulose membranes, all strips were blocked in 5% skimmed milk for 1 h at room temperature. Next, blots were incubated with the following primary antibodies at 4 °C overnight with gently shaking: METTL3, ALKBH5 and MTTL14 (1:1000; Abcam, Cambridge, MA, USA), FTO and β-actin (1:1000; Santa Cruz Biotechnology, USA), YTHDF2 (1:1000, Millipore, Bedford, MA, USA). Subsequently, the membranes were incubated with the corresponding secondary antibody (1: 5000 dilution) (Thermo Fisher Scientific, Rockford, IL, USA) for 1 h at room temperature. Finally, proteins on membrane were developed by the ECL Plus chemiluminescence detection kit (Applygen Technologies, Beijing, China) and analyzed by Image Processing Software (Image Pro Plus 6.0) (Rockville, MD, USA).