B900620

The main reagents used in this experiment include PF-543 citrate (SPHK1 inhibitor; MedChemExpress, HY-15425A, Monmouth Junction, NJ, USA), PD98059 (MEK1/2 inhibitor; MedChemExpress, HY-12028, Monmouth Junction, NJ, USA), S1P (Cayman chemical, 9002921, Ann Arbor, MI, USA), TY-52156 (S1PR3 antagonist; Cayman chemical, 19119, Ann Arbor, MI, USA), wortmannin (PI3K/Akt antagonist; Cayman chemical, 10010591, Ann Arbor, MI, USA). The specific primary antibodies include against SPHK1 (1:1000, CST, 12071S, Danvers, MA, USA), PBX1 (1:1000, CST, 4342S, Danvers, MA, USA), Phospho-Akt (Ser 473; 1:1000, CST, 4060S, Danvers, MA, USA), Akt (1:1000, CST, 4685S, Danvers, MA, USA), Phospho-p44/42 MAPK (Thr202/Tyr204; 1:1000, CST, 4370S, Danvers, MA, USA), S1PR3 (1:1000, Abcam, ab126622, Cambridge, UK), S1PR1 (1:1000, Abcam, ab23386, Cambridge, UK), CDK4 (1:1000, Santa Cruz Biotechnology, sc-23896, Dallas, TX, USA), CDK2 (1:1000, Santa Cruz Biotechnology, sc-6248, Dallas, TX, USA), CDK1/CDK2 (1:1000, Santa Cruz Biotechnology, sc-53219, Dallas, TX, USA), CyclinD1 (1:1000, Santa Cruz Biotechnology, sc-8396, Dallas, TX, USA), β-actin (1:1000, Santa Cruz Biotechnology, sc-47778, Dallas, TX, USA), goat anti-rabbit and anti-mouse horseradish peroxidases (HRPs; 1:5000, Proteintech, B900610 and B900620, Chicago, IL, USA).

RIP was performed using Magna RIP RNA‐Binding Protein Immunoprecipitation Kit (17‐700, Sigma‐Aldrich) according to the manufacturer's protocol. Briefly, UMUC3 cells with stable expression of HA‐G3BP1, SLU7‐Flag, or PABPC1‐GFP were lysed using RIP lysis buffer supplemented with a protease inhibitor cocktail and RNase inhibitor. Cell lysates were then incubated overnight at 4 °C under gentle rotation with antibodies against HA (26183, Thermo Fisher Scientific), Flag (8146, Cell Signaling Technology), GFP (66002‐1‐Ig, ProteinTech), or mouse IgG (B900620, ProteinTech). Each mixture was then incubated with protein A/G magnetic beads for 2 h at 4 °C under gentle rotation, and the immunoprecipitants were washed eight times with RIP wash buffer supplemented with a protease inhibitor cocktail and RNase inhibitor. Finally, total RNA was extracted using TRIzol reagent, after which RNA‐seq or qRT–PCR analysis were performed.
RIP‐seq was performed by RiboBio using the Illumina NovaSeq 6000 System. Ribosomal RNA sequences were removed. Twelve gigabytes of clean data per sample were obtained using RIP‐seq, and the clean reads were aligned to the human genome (hg19) using TopHat (version 2.0.13) software. Fisher's exact test was used to determine the statistical difference between HA‐G3BP1‐immunoprecipitated and input samples.

The coimmunoprecipitation method was used to examine the interaction between TRIM29 and mutant P53 protein in HT29 cells. Briefly, 800 μl IP lysis solution was first added to the cell pellet and lyse for 30 minutes. It was centrifuged at 12,000 rpm for 15 minutes at 4°C. The centrifuged supernatant was transferred to a new 1.5 ml centrifuge tube, added with 2 μg normal mouse lgG (B900620, Proteintech), 2 μg normal rabbit lgG (B900610, Proteintech), mouse-derived P53 antibody, and rabbit-derived TRIM29 antibody. The mixture was incubated overnight at 4°C. 20 μl protein A/G agarose beads were added and mixed with 200 μl IP lysate. The cell lysate was added and incubated with antibody overnight to the pretreated protein A/G agarose beads. After coimmunoprecipitation, the mixture was centrifuged at 3000 rpm for 3 min at 4°C. The supernatant was carefully removed, and placed in a new 1.5 ml centrifuge tube. The agarose beads were washed with 400 μl IP lysis solution 4 times. The precipitate was collected. The supernatant was retained.