Anti flag hrp

Cells were lysed in RIPA Buffer (Sigma) supplemented with protease inhibitor cocktail (Sigma). Protein concentration was measured using BCA protein assay reagent (Thermo Scientific) and BioTek Synergy 2 Multi-Mode Microplate Reader. Lysates were mixed with loading buffer and boiled for 5 min; twenty-five micrograms of protein were run in NuPage 4–12% Bis-Tris Gel polyacrylamide gels (Bio-Rad) and transferred to nitrocellulose membranes. Nonspecific antibody binding was blocked with TBS-T (50 mM Tris, 150 mM NaCl and 0.1% Tween-20) with 5% nonfat milk for 1 h at room temperature. The membranes were incubated with primary antibodies: anti-FLAG-HRP (1:1000, Cell Signaling 2044) in 5% milk in TBS-T for 60 min at room temperature; anti-GAPDH (1:5000, Cell Signaling, clone 14C10) in 5% milk in TBS-T for 30 min at room temperature. Membranes were then washed three times with TBS-T for 15 min total. Membranes were incubated with anti-rabbit horseradish peroxidase (HRP)-conjugated antibody (Sigma, A 6154) or anti-mouse HRP-conjugated antibody (Santa Cruz, SC-2005) diluted 1:5000 for 30 min and washed with TBS-T three times for 15 min each. Membranes were visualized using the ImmunStar WesternC Chemiluminescence Kit (Bio-Rad) and images were captured using a ChemiDoc XRS+ System and processed using ImageLab software (Bio-Rad).

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.