Anti mettl3 antibody

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).

Western blotting experiments were performed in accordance with our previous study [29 (link)]. Briefly, tissues were harvested and lysed in RIPA buffer on ice, and the extracted protein was quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Next, proteins were resolved using 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, USA). Nonspecific binding sites were then blocked by immersing the membranes in 5% bovine serum albumin in PBS at room temperature. Membranes were then incubated with the following high affinity primary antibodies: anti-WTAP antibody (1:1000, Cell Signaling Technology, USA), anti-METTL3 antibody (1:1000, Cell Signaling Technology, USA), anti-METTL14 antibody (1:1000, Cell Signaling Technology, USA), and anti-GAPDH antibody (1:1000, Cell Signaling Technology, USA). After washing, membranes were incubated with peroxidase (HRP)-conjugated secondary antibody (1:1000, Cell Signaling Technology, USA). Finally, protein signals were detected using a chemiluminescence imaging analysis system (Tanon, Shanghai, China).