Rabbit anti gapdh

Co-IP was performed by Cloudseq Biotech. The cells were lysed to extract protein by Radio Immunoprecipitation Assay Lysis Buffer (Beyotime) and quantified by an enhanced BCA Protein Assay Kit (Beyotime). Supernatant (20 μl) from the cell lysis was boiled with 5× loading buffer (20 μl) was used as input control for Co-IP. The protein samples were immunoprecipitated with magnetic beads precoated with anti-EIF3A (Huabio) and anti-METTL3 (Proteintech, China) antibody. After that, the proteins binding with beads were eluted and used as the IP group for Western blotting. The protein bands were immunoblotted with specific primary antibodies: rabbit anti-NOP2 (1:1000 dilution, Huabio) and rabbit anti-GAPDH (1:1000 dilution, Huabio). After incubation with goat anti-rabbit secondary antibody (1:1000 dilution, SAB), the membranes were scanned using Bio-rad ChemiDoc MP.

Western blotting was used to confirm expression of osteogenesis-related genes (ALP, COL1, and RUNX2) and overexpression of METTL3 protein. Total proteins were extracted by Radio Immunoprecipitation Assay Lysis Buffer (Beyotime, China) and quantified by an enhanced BCA Protein Assay Kit (Beyotime). Equal amounts of protein (30 µg) were resolved through 10% SDS-PAGE (Epizyme Biotech, China) before they were transferred to polyvinylidene difluoride membranes (Millipore, U.S.A.). Bovine serum albumin (5%; BIOFROXX, German) was used to block membranes. The protein bands were immunoblotted with specific primary antibodies: rabbit anti-ALP (1:1000 dilution, Huabio, China), rabbit anti-COL1 (1:1000 dilution, Huabio, China), rabbit anti-RUNX2 (1:1000 dilution, Huabio), rabbit anti-METTL3 (1:1000 dilution, Huabio), and rabbit anti- GAPDH (1:1000 dilution, Huabio). After incubation with goat anti-rabbit secondary antibody (1:1000 dilution, SAB), the membranes were scanned using Bio-Rad ChemiDoc MP.

Capillary western blot analyses were carried out using the chemiluminescent and fluorescent western blotting Jess system with a 12–230 kDa Jess separation module (ProteinSimple, Santa Clara, CA, USA) in accordance with the manufacturer's instructions. Protein concentration was measured using the BCA method (Vazyme, Nanjing, Jiangsu, CN). Protein samples were mixed with 0.1× sample buffer and 5× master mix (ProteinSimple) to achieve a final protein concentration of 1 µg µL⁻¹ and then denatured at 95 °C for 5 min. Then, the denatured protein samples, blocking buffer, primary antibodies, HRP‐conjugated secondary antibodies, wash buffer, and chemiluminescent substrate (1:1 luminol‐peroxidase mixture) were added to specific wells of the assay plate. Rabbit anti‐YBX1 (1:10; Cell Signaling Technology, Boston, MA, USA), rabbit anti‐SHANK3 (1:10; Cell Signaling Technology), and rabbit anti‐GAPDH (1:50; HuaBio, Hangzhou, Zhejiang, CN) antibodies were used as primary antibodies. Anti‐rabbit antibodies (ProteinSimple) were used as secondary antibodies. After loading the plate, separation electrophoresis was performed in the capillary system and immunodetection was fully automated. The relative expression of proteins was automatically calculated using Compass for Simple Western software (version 5.0; ProteinSimple). All experiments were repeated three times.