Ab177477

RIP experiments were performed using the Magna RIP RNA-binding protein immunoprecipitation kit (Millipore, Billerica, MA, USA) and the m⁶A antibody (Abcam, Cambridge, MA, USA) following the manufacturer’s protocol.
Briefly, cells (1×10⁷) were lysed with ice-cold RIP Lysis Buffer. Collect and store the cell lysate at -80 °C. Prepare the magnetic bead-antibody complex with 5 μg of the above-mentioned target antibody or control IgG and 50 μl of protein A/G magnetic beads, rotating at room temperature for 30 min. Take an equal volume of cell lysate and incubate the magnetic bead-antibody complex with rotation at 4 °C overnight so that the antibody can fully contact and bind to the protein. At the same time,10 μl cell lysate was extracted and used as input. Next day, RNA was extracted and purified with the prepared proteinase K buffer. Acquired RNA was used as a template to synthesize the corresponding cDNA.
Co-precipitated RNAs used for the first strand cDNA synthesis with the Reverse Transcription System (Roche) following the manufacturer’s protocol. Acquired cDNA was then analyzed by RT-qPCR. The information of IGF2BP1/2/3 antibody are ab184305, ab117809 and ab177477 (Abcam, Cambridge, MA, USA).

Proteins were extracted using radioimmunoprecipitation assay buffer (ab288006; Abcam) and quantified by BCA Protein Assay Kit (PICPI23223; Thermo Fisher Scientific). 40 μg of protein was separated by sodium dodecyl-sulfate–polyacrylamide gel electrophoresis and then transferred to polyvinylidene fluoride membranes. After blocking, membranes were incubated with antibodies against PIGT (1:500; 16,906–1-AP; Proteintech Group, Inc., Rosemont, IL, USA), GLUT1 (1:30,000; ab115730; Abcam), METTL3 (1:1000; ab195352; Abcam), METTL14 (1:500; ab220030; Abcam), WTAP (1:1000; ab195380; Abcam), IGF2BP1 (1:1000; ab184305; Abcam), IGF2BP2 (1:1000; ab129071; Abcam), IGF2BP3 (1:1000; ab177477; Abcam), and β-actin (1:1000; ab8226; Abcam) at room temperature for 1 h. Secondary antibodies were labeled with HRP (1:1000; A0208, A0216; Beyotime Biotechnology, Shanghai, China). Specific signals were visualized using an enhanced chemiluminescence substrate kit (P0018F, Beyotime Biotechnology).

Antibodies used in this study were: anti-Myc tag (ab32, Abcam), anti-IGF2BP1 (ab290736, Abcam), anti-IGF2BP2 (ab128175, Abcam), anti-IGF2BP3 (ab177477, Abcam), anti-RBM15 (10587-1-AP, Proteintech), anti-DNMT1 (ab92314, Abcam), anti-DNMT3A (ab307503, Abcam), anti-DNMT3B(67,259, Cell Signaling Technology), anti-SEMA3F (SAB2107196, Sigma), anti-S9.6 (MABE1095, Sigma), anti-5-mC (ab214727, Abcam), anti-m⁶A (SAB5600251, Sigma), anti-YAP1(phosphor S127, ab76252, Abcam), anti-YAP1(66900-1-Ig, Proteintech), anti-LATS1(66569-1-Ig, Proteintech), anti-LATS2(20276-1-AP, Proteintech), anti-β-Actin(4970, Cell Signaling Technology). siRNAs against DNMT1 used were purchased from Ribobio Co.,Ltd (Guangzhou, China). pcDNA3-based vectors encoding wild-type, RRM domain mutant, KH domain mutant Myc-tagged IGF2BP1, IGF2BP2, IGF2BP3 were produced by Shanghai Yoche Biotechnology Co.,Ltd (Shanghai, China). The plasmids encoding RBM15 and SEMA3F (h-RBM15-pcDNA3.1-c-HA, M35-FLAG-SEMA3F) were obtained from Guangzhou FulenGen Co., Ltd (Guangzhou, China). DNA and RNA oligos synthesized by TsingKe Biotech Co., Ltd (Beijing, China) are listed in Supplementary Table S1.

Cell lysates were obtained using radioimmunoprecipitation assay lysis buffer (Beyotime, Shanghai, China) and measured via a bicinchoninic acid Protein Assay Kit (Beyotime). Approximately 20 µg of lysate was loaded per well on 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis gels followed by electronic transfer onto polyvinylidene fluoride membranes (Millipore), and 5% fat-free milk was used for blocking followed an overnight incubation at 4 ℃ with primary antibodies against METTL3 (ab195352; Abcam), METTL14 (ab220030; Abcam), WTAP (ab195380; Abcam), CD9 (ab223052; Abcam), CD63 (ab216130; Abcam), TSG101 (ab125011; Abcam), NLRP3 (ab263899; Abcam), caspase-1 (ab207802; Abcam), gasdermin-N domain (GSDMD-N; ab215203; Abcam), IGF2BP1 (ab82968; Abcam), IGF2BP2 (ab129071; Abcam), IGF2BP3 (ab177477; Abcam), and GAPDH (#5174, Cell Signaling Technology, Danvers, MA, USA). After antibody labeling, membranes were washed with Tris-Buffered Saline Tween-20 thrice times in total. Membranes were then hybridized with horseradish peroxidase-conjugated secondary antibodies (A0208, A0216; Beyotime). Protein was identified using an Enhanced Chemiluminescence Detection kit (Pierce Biotechnology, Rockford, IL, USA) and band illumination was quantified using Image-Pro Plus 6.0 software. GAPDH was used as a loading control.

After the total protein was extracted, the protein concentration was measured with the BCA protein assay kit (Beyotime Biotechnology, China). Protein samples were electrophoresed, transferred to PVDF membranes, and blocked with 5% nonfat milk at RT for 1 h. Then the PVDF membrane was incubated with the primary antibody solution anti-RBM15 antibody (ab244374; Abcam, Shanghai, China, 1:1000), anti-TMBIM6 antibody (ab18852; Abcam, Shanghai, China, 1:1000) and anti-IGF2BP3 antibody (ab177477; Abcam, Shanghai, China, 1:1000) overnight at 4 °C. Monoclonal anti-rabbit IgG (RS0002; Immunoway, Plano, USA, 1:5000) or monoclonal anti-mouse IgG (3420; Abcam, Shanghai, China, 1:5000) were incubated with the PVDF membrane for a secondary step 1 h at room temperature. Finally, chemiluminescence (ECL) detection reagents (Beyotime Biotechnology, China) were prepared under dark conditions to detect blots.