E3f2a

Total protein was first extracted using Western and IP cell lysis buffer (Beyotime, P0013). BCA protein assay kit (Zhonghui Hecai, Shaanxi, China) was used to determine the protein concentration obtained, and 30 μg of total protein was obtained. Then, electrophoresis was performed according to previous studies [25 (link)], and PVDF membranes (Millipore, Billerica, Massachusetts, USA) were used for protein transformation. Subsequently, primary antibodies against METTL3 (monoclonal) (1 : 1,000, E3F2A, Cell Signaling), Runx2 (monoclonal) (1 : 1,000, ab236639, Abcam), and GAPDH (monoclonal) (1 : 1,000, D11H16, Cell Signaling) were incubated with membranes. TBST is used to wash the membranes, and then incubated membranes with the corresponding secondary antibody (polyclonal) (1 : 10,000, Invitrogen, USA) for 1 hr. ImageJ software (V1.8.0) was used for quantitative analysis.

RIPA lysis buffer (Beyotime, Shanghai, China) was used to lyse the cells on ice which were then centrifuged 12,000× g at 4 °C for 15 min. The BCA protein determination kit (Beyotime, Shanghai, China) was used to determine the protein content. The proteins were isolated by 10% SDS-PAGE and transferred to PVDF membranes (Merck-Millipore, Darmstadt, Germany). Membranes were blocked with 5% skimmed milk (Beyotime, Shanghai, China) for 1 h and probed overnight with anti-METTL3 antibody (1:1000, Cat.#E3F2A, Cell Signaling Technology, Danvers, MA, USA) and anti-GAPDH antibody (1:1000, Cat.#AC001, Cell Signaling Technology, Danvers, MA, USA) at 4 °C overnight, and followed by incubation with HRP-conjugated secondary anti- IgG antibody (1:1000, Beyotime, Shanghai, China) for 1 h at room temperature. Membranes were detected with HRP substrate luminol reagents (Merck-Millipore, Darmstadt, Germany), and scanned using the Gel Doc EZ Imager (Bio-Rad, Berkeley, CA, USA). Quantification of blot intensity was performed using ImageJ software (V1.8.0, NIH, MD).