m6A Dot Blot Assay

Dot blot was performed as previously described with minor modification (Chen et al., 2015). Briefly, appropriate number of cells was and harvested avoiding RNA degradation, then purified by Dynabeads mRNA purification kit (Invitrogen, #61012). RNA samples were quantified and denatured at 95°C for 3 min, then cooled down on ice for 2 min. Samples were spotted on the membrane (Amersham Hybond-N⁺, GE) and air dry for 5 min, followed by UV-crosslink (2 auto-crosslink, 150 mJ/cm² UV Stratalinker, STRATAGENE). Membranes were washed in TBST and dyed in Methylene-blue (Sigma-Aldrich) as quantitative control. Then blocked in 5% non-fat dry milk in TBST for 2 h at room temperature, incubated with m⁶A antibodies (1:1,000, Synaptic Systems, 202-003) for overnight at 4°C. After 3 time washes, membranes were incubated with HRP linked secondary anti-rabbit IgG antibody (1:5,000, CST, #7074) for 1 h at room temperature. Signals were detected with ECL Plus Western Blotting Reagent Pack (GE Healthcare). The dots were quantified by Image J.