Ythdc1

The Magna RIPTM RNA-binding protein immunoprecipitation kit (Millipore, USA) was used to carry out the RIP assay in accordance with the protocol. Magnetic beads were coated with antibodies against IgG (Millipore, MA, USA), HOXC8 (Proteintech, Wuhan, China), AGO2 (Abcam, Burlingame, USA), PD-L1 (Abcam, Burlingame, USA), METTL3, and YTHDC1 (Cell Signalling Technology, MA, USA) for 30 minutes at room temperature. To capture circSLCO1B3, the cell lysate from 2×10⁷ cells was incubated with magnetic beads coated with antibodies. TRIzol was utilized to extract the entire RNA for MeRIP. In a manner similar to RIP, magnetic beads from Millipore in Massachusetts, USA were coated with 5 μg of an anti-IgG or anti-m6A antibody and incubated at room temperature for 30 minutes. Following this, 50μg of overall RNA was introduced to the beads coated with antibodies, and then incubated in immunoprecipitation buffer containing RNase inhibitor at a temperature of 4℃ for the duration of the night. Following the digestion of proteinase K, the m6A-bound RNA precipitation was completed using the RNA Clean & Concentrator kit (ZYMO research, Hangzhou). The m6A enrichment of circSLCO1B3 was assessed using qPCR and normalized to the input.

Tissue or cell samples were lysed in RIPA lysis buffer, and the supernatant was collected by centrifugation. The protein lysate was boiled after adding the loading buffer. Protein samples were then separated by 10% sodium dodecyl sulfate-polyacrylamide (SDS–PAGE) gels and subsequently transferred to PVDF membranes. The membranes were blocked with 5% BSA. Primary antibodies were incubated overnight followed by a one-hour incubation with HRP-conjugated secondary antibodies. Protein detection was achieved using the ECL L detection system. The following antibodies were used in the experiment: GAPDH (Proteintech, 10494-1-AP), YTHDC1 (Cell Signaling Technology, 54737S), BMI1 (Abcam, ab269678), SOX2 (Abcam, ab97959), OCT4 (Abcam, ab18976), and NANOG (Abcam, ab109250).

Cultured cells were lysed and protein was extracted using radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with protease inhibitor (CW2200S, CWBio, China) and phosphatase inhibitors (CW2383S, CWBio, China). Equal amounts of proteins were resolved by 10% or 6% SDS-PAGE and were subjected to immunoblot analysis using standard methods with the following antibodies: NHPS2 (ab50339, Abcam), Synaptopodin (21064-1-AP), METTL3 (96391 s, Cell Signaling), YTHDC1 (77422 s, Cell Signaling), ADAR1(bs-2168R, Bioss), DNMT1 (ab188453, Abcam), WTAP (60,188–1-Ig, Proteintech) (all used at a 1:1000) and β-actin(AP0060, Bioworld) (used at 1:5000). The Secondary antibodies were purchased from Dingguo Biotechnology (Beijing, China). Semiquantitative analysis of the protein density by western blotting was performed using ImageJ (Version 1.5.3).