Western stripping buffer

F12-K nutrient mixture (Kaighn’s modification) and fetal bovine serum (FBS) were purchased from GIBCO Co. (NJ, USA). Glutamine, penicillin, streptomycin and Lipofectamine™ 2000 were purchased from Invitrogen (CA, USA). Quartz filters (203 × 254 mm) were purchased from Whatman Co. (PA, USA). Hoechst 33342 dye, thiazolyl blue tetrazolium bromide (MTT), pyrrolidine dithiocarbamate (PDTC) and lipopolysaccharide (LPS) (E coli, serotype 055:B5) were purchased from Sigma Chemical Co. (MO, USA). The Annexin V-FITC apoptosis detection kit was purchased from Biosea Biotechnology Co. (Beijing, China). PVDF membranes were purchased from Millipore Co. (MA, USA). Anti-β-actin, anti-Bad (19–35) rabbit pAb were purchased from MBL (MA, USA). The Phototope®-HRP western blot detection system was purchased from Cell Signaling Co. (MA, USA). Western stripping buffer was purchased from the Beyotime institute of biotechnology (Beijing, China). The Cell and tissue protein extraction reagent, protease inhibitor cocktail, phosphotase inhibitor cocktail and phenylmethylsulfonyl fluoride (PMSF) were purchased from KangChen Biotech Co (Beijing, China). All other chemicals were analytical grade and were purchased from Sigma Chemical Co. or DingGuo Biochemical Co. (Beijing, China).

Fresh purified NK cells were lysed in M-PER Mammalian Protein Extraction Reagent (Thermo). Then, samples were run on precast 4–12% Bis–Tris protein gels (Genscript). Separated proteins were transferred to polyvinylidene fluoride (PVDF) membranes and blocked with 5% w/v skimmed milk at room temperature for 1 h. Then, PVDF membranes were incubated with primary antibodies in 5% w/v skimmed milk in Tris-buffered saline containing 0.1% Tween-20 at 4 °C overnight, then incubated with Anti-rabbit IgG, HRP-linked Ab (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature. To detect several proteins on the same PVDF membrane, membranes were processed with western stripping buffer (Beyotime) and reincubated with primary Abs. Chemiluminescence autoradiography was used to detect protein bands. The primary Abs for METTL3, WTAP, YTHDC2, YTHDC1, YTHDF1, ALKBH5, FTO, SHP2, AKT, p-AKT S473, STAT5, p-STAT5 Y694, Lamin B1, mTOR, p-mTOR Ser2448, p38 MAPK, p-p38 MAPK Thr180/Tyr182, and β-actin were purchased from Cell Signaling Technology; primary Ab SOCS3 was purchased from Abcam. The Anti-rabbit IgG, HRP-linker antibody was purchased from Cell Signaling Technology. The dilution of all antibodies used for immunoblotting was 1:1000.