Mettl16

The total protein was extracted using a RIPA kit (Huaxing Bio, Beijing, China), and then the protein concentration was quantified using a bicinchoninic acid (BCA) kit (Thermo Fisher). Protein samples (25 ug) were separated on electrophoresed in polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, MA, United States). After blocking in 5% skimmed milk at room temperature for 1 h, membranes were incubated with primary antibodies against FTO (Proteintech, Wuhan, China), METTL16 (Proteintech), YTHDF2 (Abcam), CBLL1 (Proteintech), and GAPDH (Huaxing bio) at 4°C overnight. The membranes were incubated with HRP-conjugated secondary antibodies (Huaxing Bio) for 1 h and visualized by an enhanced chemiluminescence blotting kit (Cwbiotech, Jiangsu, China). The intensities of the bands were quantified using Quantity One software (Bio-Rad, CA, United States). GAPDH was used as the internal control.

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.