Rabbit anti mettl3

Human OS tissue and cell lines were lysed in RIPA lysis buffer (Cat# P0013B, Beyotime) supplemented with a 1× protease inhibitor cocktail (Cat# P1005, Beyotime) for 30 min at 4°C, followed by a 15-min centrifugation step at 12,000 × g to generate supernatants. Then, a bicinchoninic acid protein assay kit (Cat# ab102536, Abcam) was used to determine protein concentrations. Next, 5× protein loading buffer (Cat# P0015; Beyotime) was added to lysates and denatured for 5 min at 100°C. Approximately 20 μg protein was separated using sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene fluoride membranes. Membranes were then blocked in 5% fat-free milk for 45 min at room temperature, followed by an overnight incubation with primary antibodies at 4°C. Next, membranes were rinsed three times in 1× TBST buffer and further incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 60 min at room temperature. Protein signals were determined using enhanced chemiluminescence reagent (Cat# K-12045-D10, Advansta, San Jose, CA, USA). Antibodies were rabbit anti-Mettl3 (Cat# 15073-1-AP, Proteintech, Wuhan, China), rabbit anti-β-actin (1:3,000, Cat# 4,970, Cell Signaling, Danvers, MA, USA), and goat anti-rabbit IgG H&L (HRP) (1:1,500, Abcam, Cat# ab205718).