Mettl3

HCC specimens were collected during surgery. The proteins separated on gels after electrophoresis are transferred to PVDF membranes and subsequently incubated with primary and secondary antibodies. The following primary antibodies were incubated for 12 h at 4 °C: GAPDH (1:10,000, ABclonal), YTHDF1 (1:1000, ABclonal), YTHDF2 (1:1000, ABclonal) and YTHDF3 (1:1000, ABclonal), KIAA1429(1:1000, Proteintech), METTL3(1:1000, Proteintech), RBM15(1:1000, Proteintech), ZC3H13(1:1000, Proteintech), METTL16(1:1000, Proteintech), ALKBH5(1:1000, Proteintech), METTL14(1:1000, Proteintech), WTAP(1:1000, Proteintech), HNRNPC(1:1000, Proteintech), YTHDC1(1:1000, Proteintech), YTHDC2(1:1000, Proteintech), FTO(1:1000, Proteintech). The secondary antibody (1:5000, ABclonal) was incubated for 2 h at room temperature. Immunoreactive bands were developed using an enhanced chemiluminescence detection kit (Genview Scientific Inc., USA). All experiments were performed in triplicate.

H9c2 cells and HUVECs were lysed using RIPA lysis buffer (Beyotime, Shanghai, China). Protein concentration was determined using the BCA Protein Assay kit (New Cell and Molecular Biotech, Suzhou, China). Specifically, the corresponding protein was separated with 7.5 and 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. Afterward, the membranes were blocked with 5% skim milk for 2 h in room temperature (Qiu et al., 2020 (link)). They were incubated with primary antibodies against GAPDH (Proteintech, Wuhan, China); Tet1 (Signalway Antibody, College Park, MD); Hadh (Signalway Antibody, College Park, MD); Kcnn1 (Signalway Antibody, College Park, MD); Caspase3 (Proteintech, Wuhan, China); and Cleavd-Caspase3 (Cell Signaling Technology, Danvers, MA); bcl-2 (Abcam, Cambridge, MA); Bax (Abcam, Cambridge, MA); HIF-1α (Abcam, Cambridge, MA); and Mettl3 (Proteintech, Wuhan, China) overnight at 4°C. Then, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Abcam, Cambridge, MA) for 1 h. The bands were analyzed with chemiluminescence Western blotting detection system (Tanon, Shanghai, China) (Gu et al., 2018 (link); Ren et al., 2018 (link); Li X. et al., 2020 (link)).

Cells were washed twice with cold PBS. Then the cells were lysed with RIPA lysis buffer (50 × 10⁻³m Tris‐HCl (pH 7.4), 150 × 10⁻³m NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% SDS, sodium orthovanadate, sodium fluoride, EDTA, leupeptin) for 30 min. The cell lysate was centrifuged at 12 000 rpm for 15 min. The protein concentration was measured using BCA protein assay kit. Equal amounts of total protein were separated on 10% or 15% SDS‐PAGE and transferred onto NC membranes, blocked with 5% nonfat milk at room temperature for 1 h, and incubated with primary antibodies at 4 ˚C overnight. After washed three times, the membranes were incubated with secondary Ig conjugated HRP for 1 h at room temperature. Protein bands were visualized with the ECL enhanced chemiluminescence regent Kit (NCM Biotech). The following antibodies were used: RBMS1 (Abcam ab150353), S100P (Proteintech 11803‐1‐AP), YTHDF1 (Proteintech 17479‐1‐AP), YTHDF2 (Proteintech 24744‐1‐AP), YTHDF3 (Proteintech 25537‐1‐AP), METTL3 (Proteintech 15073‐1‐AP), METTL14 (Proteintech 26158‐1‐AP), FTO (Proteintech 27226‐1‐AP), m6A (Proteintech 68055‐1‐lg), FLAG (Sigma 1804), V5 (Proteintech 14440‐1‐AP), VINCULIN (Proteintech 66305‐1‐Ig), GAPDH (Proteintech 60004‐1‐Ig).