Enhanced chemiluminescence blotting kit

After MC3T3-E1 cells were cultured in OM for 7 days, total protein was extracted and quantified with the method described in the ALP activity assay. Samples with equal amounts of denatured protein were analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. Proteins were transferred to polyvinylidene difluoride membranes (Millipore, Bedford, MA, United States) and blocked in 5% skimmed milk. Then, membranes were incubated with primary antibodies against RUNX2 (CST, Danvers, MA, United States), BSP (CST), OSX (Abcam, Cambridge, United Kingdom), and GAPDH (Huaxing bio) at 4°C overnight. After washing in TBS-T, the membranes were incubated with HRP-conjugated secondary antibody (Huaxing bio) for 1 h at room temperature and visualized using an enhanced chemiluminescence blotting kit (Cwbiotech).

The total protein was extracted using a RIPA kit (Huaxing Bio, Beijing, China), and then the protein concentration was quantified using a bicinchoninic acid (BCA) kit (Thermo Fisher). Protein samples (25 ug) were separated on electrophoresed in polyacrylamide gels and transferred onto polyvinylidene difluoride membranes (Millipore, MA, United States). After blocking in 5% skimmed milk at room temperature for 1 h, membranes were incubated with primary antibodies against FTO (Proteintech, Wuhan, China), METTL16 (Proteintech), YTHDF2 (Abcam), CBLL1 (Proteintech), and GAPDH (Huaxing bio) at 4°C overnight. The membranes were incubated with HRP-conjugated secondary antibodies (Huaxing Bio) for 1 h and visualized by an enhanced chemiluminescence blotting kit (Cwbiotech, Jiangsu, China). The intensities of the bands were quantified using Quantity One software (Bio-Rad, CA, United States). GAPDH was used as the internal control.