M6a antibody

Total RNA was separately extracted from human tissues using TRIzol (Invitrogen) reagent and the RNA concentration was determined using a NanoDrop 2000 (Thermo Fisher Scientific). The RNA concentration was then adjusted to 250, 500, and 1000 ng/μL. Two microliters of Poly(A) + RNAs were first denatured by heating at 65 °C for 5 min and spotted onto a nitrocellulose membrane (Amersham, GE Healthcare, USA). Next, the membrane was cross-linked by 1500 J UV, blocked with 5% non-fat milk twice, and incubated with m6A antibody (1:1000, Thermo Fisher Scientific, USA) overnight at 4 °C. Subsequently, the membrane was incubated with HRP-conjugated goat anti-mouse IgG dilution (1:3000, Proteintech, USA). Finally, the membrane was visualized using the chemiluminescence imaging analysis system (Tanon, Shanghai, China). The other membrane was dyed in 0.02% methylene blue (MB) for 30 min, followed by rinsing twice with ultrapure water and photographing. The membrane was visualized with chemiluminescence image analysis system (Tanon, Shanghai, China) and the grayscale was measured by ImageJ (NIH, Bethesda, MD) software.

Antibodies against the following proteins were used for Western blot: RPS15 (1:2000, Abcam; ab157193), IGF2BP1 (1:1000, Cell Signaling Technology; 8482), phospho-p38 MAPK (1:1000, Cell Signaling Technology; 9211), p38 MAPK (1:1000, Cell Signaling Technology; 9218), MKK6 (1:1000, Cell Signaling Technology; 8550), β-actin (1:1000, Cell Signaling Technology; 4970), Flag-tag (1:5000, Immunoway; YM3001), Myc-tag (1:5000, Immunoway; YM3203), m⁶A antibody (Thermo; MA5-33030) and rabbit IgG (Millipore; PP64B)-Diquafosol tetrasodium (CAS: 211427-08-6; T7423), Coenzyme A (CAS: 85-61-0; T10857), Folic acid (CAS: 59-30-3; T0062), Epicatechin gallate (CAS: 1257-08-5; T2732), and Methotrexate (CAS: 59-05-2; T1485) were purchased from TargetMol and SB203580 (CAS: 152121-47-6; T1764).