Monoclonal anti mouse igg

Cells were lysed in cell lysis buffer (Cat# P0013B; Beyotime Biotechnology, Shanghai, China) supplemented with PMSF protease inhibitor on ice for 1 h, followed by centrifuging at 13,500 g at 4°C for 15 min. Protein concentration was measured using a BCA Protein Assay Kit (Cat# P0010S; Beyotime Biotechnology) following the manufacturer’s instructions. Protein samples (50 µg) were separated on a polyacrylamide gel and transferred to a nitrocellulose membrane and then blocked with 10% fat-free dry milk at room temperature for 1 h. Then, the membrane was incubated with a rabbit anti-METTL14 antibody (1:1000; Cat# 51104S; Cell Signaling Technology), a rabbit anti-caspase-3 antibody (1:1000; Cat# 9662S; Cell Signaling Technology), and a mouse anti-GAPDH antibody (1:500; Cat# abs830030; Absin) at 4°C overnight. Then, the membrane was incubated with monoclonal anti-rabbit IgG (1:5000; Cat# ab97051; Abcam) or monoclonal anti-mouse IgG (1:5000; Cat# ab6789; Abcam) at room temperature for 1 h. Western blot bands were imaged with Odyssey CLx and quantified with LI-COR Image Studio Software (LI-COR Biosciences, Lincoln, NE, USA).

BMSCs were lysed in cell lysis buffer (catalog #P0013B; Beyotime Biotechnology, Shanghai, China) supplemented with PMSF protease inhibitor on ice for 30 min, followed by centrifugation at 13,500 × g at 4°C for 15 min. The protein concentration was quantified using a bicinchoninic acid (BCA) protein assay kit (catalog #P0010S; Beyotime Biotechnology) following the manufacturer’s instructions. Protein samples (50 μg) were separated on polyacrylamide gel, transferred onto a nitrocellulose membrane, and then blocked with 5% fat-free dry milk at RT for 1 h. Next, the membrane was incubated with rabbit anti-RUNX2 antibody (1:1,000; catalog #8486; Cell Signaling Technology, Danvers, MA, USA), rabbit anti-METTL3 antibody (1:1,000; catalog #A8370; ABclonal, Woburn, MA, USA), mouse anti-tubulin antibody (1:1,000; catalog #abs830032; Absin, Shanghai, China), or mouse anti-β-actin antibody (1:1,000, catalog #sc-47778; Santa Cruz Biotechnology) at 4°C overnight. A secondary incubation step was carried out with monoclonal anti-rabbit IgG (1:5,000, catalog #14708; Abcam) or monoclonal anti-mouse IgG (1:5,000, catalog #3420; Abcam) at RT for 1 h. Western blot bands were imaged by Odyssey CLx and quantified with LI-COR Image Studio software (LI-COR Biosciences, Lincoln, NE, USA).