Anti runx2

Total protein was extracted from cells by radioimmunoprecipitation assay buffer containing protease inhibitors. The protein concentration was detected using a BCA protein assay (BioRad, USA). Then, equal amounts of protein were loaded and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and then transferred onto nitrocellulose membranes. After blocking with 5% non-fat milk, the membranes were incubated overnight at 4°C with the following specific primary antibodies: anti-SOX5, anti-Collagen I, anti-Runx2, anti-Osterix, and anti-KLF4 (Sigma-Aldrich, USA), and anti-β-actin antibody (Sigma, USA). After incubation with a peroxidase-conjugated secondary antibodies (Sigma) for 2 h at room temperature, the target proteins were visualized using enhanced chemiluminescence reagents (Pierce, USA) on Image Reader LAS-3000 Fujifilm. The density of the bands on the membrane were quantified with Image J software. β-actin was used as an internal control.

After the soft tissue on the tibia of mice had been removed, tibias were cut by ophthalmic scissors and 80g weighed by electronic scale. The tibia of each sample was placed in the grinding tube, to which had been added grinding beads and lysis buffer, and placed in the tissue homogenizer for grinding. The supernatant and was gotten for examining concentration of total protein by bicinchoninic acid method(BCA). Polyacrylamide gel electrophoresis was performed to separate the target proteins from the samples with the equal amount of total protein. Proteins were transferred to PVDF membrane (Invitrogen) and closed non-specific binding with 5% skimmed milk. The protein was added with primal antibodies of anti-PI3K (1:1000, Abcam), anti-Akt (1:1000, Abcam), anti-pAkt (1:1000, Abcam), anti-mTOR (1:1000, Sigma), anti-Beclin-1 (1:1000, Proteintech), anti-LC3 II (1:1000, Proteintech), anti-p62 (1:1000, Proteintech), anti-NF-κB (1:1000, Proteintech), anti-Runx2 (1:1000, Sigma), anti-beta actin (1:1000, Abcam) and secondary antibodies (Abcam) in turn, and then washed with tris-buffered saline Tween (TBST). The PVDF film was developed and photographed by Alpha gel imaging system. Beta-actin was used as an internal reference, and the data were analyzed by self-contained software.