Sc 365765

Cells with different treatments or bone tissues were harvested for Western blot analysis and lysed with 50 µL of RIPA (radioimmunoprecipitation assay) buffer. After three times of ultrasound, the protein supernatant was extracted by centrifugation at 12 000 g for 15 minutes, and the protein concentration was determined by the BCA kit. The loading quantity of protein sample is 30 µg, and the loading volume is 20 µL. After completion, SDS-PAGE electrophoresis was performed and transferred to the nitrocellulose filter membrane. Then it was blocked with 5% non-fat dry milk in a shaker for 1 hour and then incubated with primary and secondary antibodies. Primary antibodies against P62 (5114), ATG5 (12994), ATG3 (3415), ATG7 (8558), ATG12 (4180), beclin-1 (3495), RUNX2 (12556) were purchased from Cell Signaling Technology. Antibody specific for LC3 (L7643) was purchased from Sigma-Aldrich. Antibodies specific for GAPDH (AC002), OCN (A1530), and β-tubulin (AC021) were purchased from ABclonal. Antibody specific for METTL14 (ab98166) was purchased from Abcam. Antibody specific for ALP (alkaline phosphatase, sc-365765) was purchased from Santa Cruz Biotechnology. Secondary antibodies were anti-mouse IgG (ImmunoWay Biotechnology, RS23910) or anti-rabbit IgG (ImmunoWay Biotechnology, RS23920). Western blot bands were imaged with Odyssey CLX and quantified with LI-COR Image Studio Software.