Mettl14

Western Blot was performed as our previously described [20 (link)]. The primary antibodies include Mettl14 (220030, Abcam, USA), FTO (27226-1-AP, Proteintech, China), α-syn (10842-1-AP, Proteintech, China), TH antibody (25859-1-AP, Proteintech, China), ATM antibody (27156-1-AP, Proteintech, China), YTHDF3 antibody (25537-1-AP, Proteintech, China), CD81 antibody (27855-1-AP, Proteintech, China), Syntenin (ab19903, Abcam, USA), HSP90B1 antibody (14700-1-AP, Proteintech, China), Calnexin antibody (10427-2-AP, Proteintech, China), TSG101 antibody (28283-1-AP, Proteintech, China), and GAPDH antibody (60004-1-AP, Proteintech, China).

The total proteins from C2C12 cells were lysed in RIPA lysis buffer supplemented with a protease inhibitor cocktail (4693124001; Roche, Mannheim, Germany). The membranes were blocked with 5% BSA for 1 h at room temperature and subsequently probed with primary antibodies overnight at 4 °C. the following dilutions were used for each antibody: METTL3, METTL14, WTAP, DNMT1, DNMT3A and DNMT3B (all 1:1000; Abcam, Cambridge, England). The membranes were then washed with PBS-Tween and incubated for 50 min with horseradish peroxidase-conjugated secondary antibodies (SA00001–1; Proteintech, Wuhan, China). Protein bands were detected after treatment with the SuperSignal west Femto agent (34094; Thermo Scientific, Waltham, USA).

BC and TNBC tissue sections were rehydrated in xylene and alcohol, and then rehydrated at 37°C with 3% H₂O₂ for 30 min. Next, all slices were incubated with goat serum for 15 min, and then washed with TBST three times. Ten minutes each time. Then, the sections were incubated overnight with METTL14 (1: 1000, Abcam) and Ki‐67 (1: 1000, Abcam) at 4°C. Next, the sections were incubated with anti‐rabbit secondary IgG antibody at 37°C for 30 min. DAB (Boster) was used for visual colour rendering of the signal.

Total protein was isolated from TNBC samples (tumour tissues, paired normal adjacent tissues and cell lines) using RIPA lysis buffer (Beyotime Institute of Biotechnology). Total protein was separated by 10% SDS‐PAGE (25 μg protein per lane), and the protein was transferred with PVDF membrane (MilliporeSigma). Blocking of PVDF membrane was performed with 5% nonfat milk powder (Beyotime). To detect the expression of PKM2 protein in different conformations, the total protein extracted from the sample was not thermal denatured and was denatured using a non‐denatured gel sample loading buffer (Beyotime). Next, protein electrophoresis was performed using native PAGE running buffer (Beyotime). The PVDF membranes were probed at 4°C overnight with antibodies against METTL14 (1:1000; Abcam), PKM2 (ProteinTech Group, Inc.), DGCR8 (1:1000; Abcam), TRIM9 (1:1000; Abcam) and β‐actin (1:5000; Abcam). Finally, protein expression was analysed by chemiluminescence reagents (Hyperfilm ECL).