Igf2bp3

Protein was extracted from GSC cells or GBM cells. Primary antibodies to the following were used: GAPDH (Cell Signaling Technology, 5174), β-actin (Cell Signaling Technology, 14074), N-cadherin (Cell Signaling Technology, 13116), E-cadherin (Proteintech, 20874-1-AP), CD44 (Proteintech, 15675-1-AP), AKT (Cell Signaling Technology, 9272), phospho-Akt (Ser473, Cell Signaling Technology, 4060), CDK1/cdc2 (Cell Signaling Technology, 9116), phospho-CDK1/cdc2 (Tyr15) (Cell Signaling Technology, 4539), cyclin B1 (Cell Signaling Technology, 12231), P21 (Cell Signaling Technology, 2947), IGF2BP3 (Abcam, ab177477), ubiquitin (Cell Signaling Technology, 3936), DYKDDDDK Tag (Cell Signaling Technology, 14793), His-tag (Cell Signaling Technology, 12698), CUL2 (Proteintech, 10981-2-AP; Santa, sc-166506), and RPN2 (Abcam, ab244399).

RIPA lysis buffer (Solarbio, Beijing, China) to extract total protein. The proteins were quantified by the bicinchoninic acid (BCA) assay kit (Solarbio, Beijing, China). Add SDS loading buffer to the extracted total protein, and then boil it in 100° water for five minutes for subsequent experiments. The obtained protein was electrophoresed in 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). PVDF membrane was used for electroporation. The protein electrophoresis was performed at a stable voltage of 100 V, and the electroporation was performed at a stable current of 300 mA for 90 min. After electroporation, soak the PVDF membrane in 5% skimmed milk and place it on a shaker for half an hour. Then add 5 ml of CCND2 (1:1000), IGF2BP3 (1:1000) (Abcam, CA, USA), RB (1:500), and GAPDH (1:5000) (Cell Signaling Technology, MA, USA) rabbit-derived primary antibody to the PVDF membrane and incubate overnight at 4°. After incubating overnight, collect the primary antibody, add Tris-Buffered Sal ine Tween 20 (TBST) and wash three times for 15 min each time. Add goat anti-rabbit (Solarbio, Beijing, China, 1:5000) and incubate for 1 h, then continue to wash with TBST three times. Finally, add electrochemiluminescence (Solarbio, Beijing, China) liquid to expose in the exposure instrument. The antibodies used in the experiments are shown in Additional file 2: Table S2.

For Western blotting, cells were lysed with RIPA lysis buffer kit (Jrdun Biotechnology, CA), supernatants were collected after spin and total proteins were measured using the BCA protein quantification kit (Thermo Scientific, USA). Total protein samples were separated by 10% or 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE). Then, the samples were transferred onto nitrocellulose membranes. After blocking with 5% fat‐free milk for 1 hour at room temperature, the membranes were incubated with primary antibodies against IGF2BP3 (1:1000; Abcam, USA), CyclinD1 (1:1000; Cell Signaling, Germany), p‐STAT3 (1:5000, Abcam, USA), STAT3 (1:2000, Abcam, USA), BAX (1:1000; Abcam, USA), Bcl2 (1:500, Abcam, USA) and GAPDH (1:2000; Cell Signaling, Germany) overnight at 4°C. Following three washes with TBST buffer, the membranes were incubated with secondary goat anti‐rabbit antibodies conjugated with HRP for 1 hour at room temperature. Signals were visualized using the enhanced chemiluminescence kit (Millipore, USA) and detected by the Tanon‐5200 Imaging system. Integrated relative densities of individual bands were quantified using Image J (National Institutes of Health, Bethesda, MD).

Total protein was extracted from cultured cells using RIPA lysis buffer containing protease inhibitors (Beyotime, Shanghai, China). Cell lysates were separated on SDS–polyacrylamide gels and electrotransferred onto polyvinylidene difluoride (PVDF) membranes that were incubated at 4 °C with primary antibodies in 5% nonfat milk in PBS containing Tween-20 (PBST). The antibodies used for this study were used at 1:1000 dilutions in primary antibody dilution buffer (Beyotime) and were as follows: GAPDH and β-actin (Beyotime), SLC7A5 (Proteintech, Chicago, USA), IGF2BP3 (Abcam, Cambridge, UK), mTOR, p-mTOR, AKT and p-AKT (Cell Signaling Technology, Beverly, MA, USA). Membranes were washed with PBST for three times and incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The blots were visualized using an ECL chemiluminescent reagent (Bioground, Chongqing, China).

RIP assay was carried out using the Magna RIP^TM RNA-Binding Protein Immunoprecipitation Kit (Millipore) as the manufacturer’s instructions. Briefly, cell lysates were incubated with magnetic beads coated with normal IgG (Millipore), RBM15 (Abcam), METTL5 (Proteintech), IGF2BP1 (Abcam), IGF2BP2 (Abcam), IGF2BP3 (Abcam), METTL3 (Cell Signaling Technology), WTAP (Abcam), YBX1 (Abcam) or GFP (Abcam). The RNA-protein complexes were washed and purified and then subjected to RNA extraction by TRIzol. The UBA6 mRNA enriched by indicated antibody was determined by qRT-PCR. MS2-RIP was performed as previous study described [22 (link)].

In human Ishikawa and HEC-1-A cells, RNA pulldown assays were conducted according to the instructions of an RNA–protein pulldown kit (NO. 20164, Thermo Scientific, Rockford, IL, USA). Biotin-labeled LINC00958 was synthesized by GenePharma (Suzhou, China) and interacted with cell lysates for 4 hours. LacZ was employed as the negative control. Streptavidin magnetic beads pulled down the proteins bonded with biotin-labeled LINC00958 or LacZ following overnight incubation. The generated protein-RNA-bead compounds were collected, and then the proteins were eluted. Finally, western blot assays were used to test the retrieved proteins. The primary antibodies used were as follows: GAPDH (AF7021, Affinity Biosciences, Changzhou, China, 1:3000), IGF2BP3 (ab179807, Abcam, USA, 1:1000).