Anti mettl3

Mouse cerebellum and cerebral cortex were dissected as described above and triturated in RIPA buffer supplemented with protease and phosphatase inhibitors; 60–80 µg of tissue lysates were subjected to SDS-PAGE and western blot analysis. Primary antibodies used in our analysis are as follows: anti-METTL3 (Abnova, H00056339-B01P), anti-METTL14 (Atlas Antibodies, HPA038002), anti-ALKBH5 (Sigma, HPA007196), anti-WTAP (Santa Cruz, sc-55438), anti-FTO (Abcam, ab92821) and beta-ACTIN (Santa Cruz, sc-47778). Secondary antibodies used are as follows: peroxidase-conjugated AffiniPure Goat Anti-Rabbit IgG (H + L) (XiYA Biology, Beijing, FZ-4201), peroxidase-conjugated AffiniPure Goat Anti-Mouse IgG (H + L) (XiYA Biology, Beijing, FZ-4202) and peroxidase-conjugated AffiniPure Rabbit Anti-Goat IgG (H + L) (XiYA Biology, Beijing, FZ-4203).

GST-fused WTAP, and its deletion mutant proteins as bait, were produced by SoluBL21-competent Escherichia coli (Genlantis). pEZ-FLAG-mMETTL3 or pEZ-FLAG-mMETTL14 was transfected into COS cells, and their lysates (prey) were incubated with GST-WTAP or its mutant proteins immobilized on glutathione-Sepharose 4B (GE Healthcare Life Sciences). After extensive washing, the protein complex was eluted with 10 mg/ml reduced glutathione and assessed by Western blotting with anti-METTL3 (Abnova), METTL14, and FLAG M2 antibody (Sigma). NP-40 (0.02%) was used for the lysis buffer, the binding buffer, and the washing buffer in this assay.