Clb buffer

Cells were washed twice with ice-cold PBS and ruptured with CLB buffer (Cell Signaling Technology) with cocktail protein inhibitor and the RNase inhibitor. After lysis, the RNA in the whole-cell lysis was fragmented into ~500 bp in length by the ultrasonic sound (1/10 volume was separated as input) and then immunoprecipitation was carried out with m⁶A, Flag, or IgG antibodies overnight at 4 °C. The precipitated RNA was extracted using Trizol reagent and was reverse transcribed with a PrimeScript RT-PCR Kit. qPCR analysis of the retrotranscribed RNA was performed with specific primers as indicated.

Cells were washed twice with ice-cold PBS and ruptured with CLB buffer (Cell Signaling Technology) containing PMSF and cocktail inhibitor. Cell lysates were resolved by SDS-PAGE and transferred onto nitrocellulose membranes and then blotted. Specific antibodies used are listed below: anti-Mettl3 (15073–1-AP, 1:1000) antibody was from Proteintech. Anti-Mettl14 antibody (HPA038002, 1:000) was from Sigma; Anti-Fto (ab124892, 1:1000), anti-Wtap (ab118339, 1:500) were from Abcam; antibody to p65 phosphorylated at Ser536 (3031S, 1:1000), antibody to IKKα-IKKβ phosphorylated at Ser176 and Ser180 (2697S, 1:1000), antibody to Erk phosphorylated at Thr202 and Tyr204 (9106S, 1:1000), antibody to Jnk phosphorylated at Thr183 and Tyr185 (9255S, 1:1000), anti-β-actin (3700S, 1:10000), anti-Flag-HRP (2044S, 1:2000), anti-p65 (6956S, 1:1000), anti-p38 (9212S, 1:1000), anti-IκBα (9242S, 1:1000), and anti-IKKβ (8943S, 1:1000) were from Cell Signaling Technology. All of the unprocessed scans of the blots were shown in the Source Data file.