Rbm15

After being washed using PBS, the cells were lysed with RIPA (radioimmunoprecipitation assay) solution containing a protease inhibitor. The acquired proteins were then quantified using a bicinchoninic acid protein assay (BCA) kit, after which 20 mg of total protein was separated on 8% or 10% sodium dodecyl sulfate polyacrylamide gel (SDS-PAGE) under electric field. The separated proteins were blotted onto a polyvinylidene difluoride (PVDF) membrane (Millipore, United States), which was then blocked with 5% bovine serum albumin (BSA) solution for 1 h and incubated with primary antibodies against GAPDH (1:1,000, Proteintech, United States), RBM15 (1:1,000, Proteintech, United States), METTL14 (1:1,000, Proteintech, United States), PDCD1 (1:2,000, Proteintech Group, United States), CTLA4 (1:1,000, novus biotechnologies, United States), HNRNRC (1:1,000, abcam, United States), and BTLA (1:1,000, abcam, United States) overnight at 4°C. On the second day, the membrane was incubated with secondary antibodies (1:2,500) at room temperature for 1 h after three times of washes with TBST (Tris-buffered saline-Tween). After being washed for another three times, bands of conjugate proteins were visualized via a ChemiDoc™ MP Imaging system (Bio-Rad Laboratories) with GADPH as the internal control.

Proteins were obtained from cells or tissues using RIPA lysis buffer. Protein concentration was measured using a BCA kit. Then, 25 μg of protein per sample was separated by SDS-PAGE., Protein was transferred to PVDF membranes. The membrane was blocked with 5 % non-fat milk for 1 h, incubated with the primary antibody overnight, and then incubated with the secondary antibody for 1 h. Finally, the membrane was incubated with ECL to observe the protein bands. The primary antibodies and secondary antibodies included UltraPolymer Goat anti-Mouse IgG (H&L)-HRP (proteintech, #PR30012), G3BP1 (proteintech, #13057-2-AP), RBM15 (proteintech, #10587-1-AP), YTHDF2 (proteintech, #24744-1-AP), MMP2 (proteintech, #10373-2-AP), NOTUM (proteintech, #14663-1-AP), CD82 (proteintech, #66803-1-Ig), GAPDH(proteintech, #10494-1-AP), UltraPolymer Goat anti-Rabbit IgG (H&L)-HRP (proteintech, #PR30011), and MMP9 (N-terminal) (proteintech, #10375-2-AP).

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.