Ab209484

Total proteins were extracted using RIPA reagent (Beyotime Biotechnology, Shanghai, China) and the protein concertation was determined by BCA kit (Beyotime Biotechnology, Shanghai, China) as per the instructions. Then, 25 μg of protein was loaded on 10% SDS-PAGE and transferred on PVDF membranes (Millipore, Billerica, MA). After blocking with 5% skimmed milk for 1 h, PVDF membranes were incubated with primary antibodies (rabbit anti-RUNX2, 1:1000, ab236639; rabbit anti-OCN, 1:1000, ab133612; rabbit anti-OSX, 1:1000, ab209484; rabbit anti-OPN, 1:1000, ab8448; rabbit anti-GAPDH, 1:1000, ab9485; all purchased from Abcam, Shanghai, China) overnight at 4°C. The following day, membranes were washed 3 times with TBST buffer for 5 minutes each, and further incubated with secondary antibody (goat anti-rabbit H&L preadsorbed, 1:1000, ab7090; purchased from Abcam, Shanghai, China) for 2 h at room temperature. The protein bands were visualized using an enhanced chemiluminescence kit (Yeasen, Shanghai, China) and photographed on a gel imager system (Bio-Rad, Hercules, CA, United States). GAPDH functioned as the internal control.

The decalcified samples were dehydrated in 15% sucrose/PBS solution for 2 h, 30% sucrose/PBS for 2 h, and 30% sucrose/OCT (4,583, Sakura, Torrance, CA, United States) overnight at 4°C and then embedded in OCT. Cryosections measuring 8 μm in thickness were immunofluorescence-stained following standard protocols. The primary antibodies included Runx2 (1:100, #12556, Cell Signaling Technology, Danvers, MA, United States), Osterix (1:100, ab209484, Abcam, Cambridge, United Kingdom), β-galactosidase (β-gal; 1:200, ab9361, Abcam), osteocalcin (Ocn; 1:100, ab93876, Abcam), and Sox9 (1:100, ab185230, Abcam). Alexa Fluor 488 and Alexa Fluor 568 (1:200, Invitrogen, Waltham, MA, United States) were used as secondary antibodies. DAPI (62248, Invitrogen) was used for counterstaining. ImageJ was used to analyzed the ratio of Osterix+/tdTomato + cells to Osterix + cells.
Cell samples were plated in a 4-well chamber slide (PEZGS0416, Millipore). The slides were washed with PBS, immediately fixed with 4% paraformaldehyde for 15 min, and blocked with goat serum for 30 min at room temperature. The cells were incubated with the primary antibody (Ki67, 1:200, ab15580) at 4°C overnight, then incubated with secondary antibodies at room temperature for 1 h, stained with phalloidin for 20 min (1:50, A22287, ThermoFisher Scientific), and counterstained with DAPI.

The extracts of total protein were prepared by lysing MC3T3-E1 cells in a RIPA buffer containing protease inhibitors. BCA assays were employed to determine the concentrations of protein. 30 μg proteins of each sample were added to 10% SDS-PAGE and then electrotransferred to the PVDF for further immunoblotting. Antibodies were used: anti-Runx2 (1:1000, ab236639, Abcam), anti-Osx (1:1000, ab209484, Abcam), anti-p53 (1:1000, 60283-2-Ig, Proteintech), anti-CyclinD1 (1:1000, 26939-1-AP, Proteintech), anti-Bcl-2 (1:1000, 26593-1-AP, Proteintech), anti-Bax (1:1000, 60267-1-Ig, Proteintech), anti-p38 (1:1000, #8690, Cell Signal Technology), anti-p-p38 (1:1000, #4511, Cell Signal Technology), anti-JNK (1:1000, #9255, Cell Signal Technology) anti-p-JNK (1:1000, #9255, Cell Signal Technology), and anti-β-actin antibody (1:1000, AF0003, Beyotime). PVDF were incubated with diluted antibodies. Goat anti-rabbit IgG H&L (HRP) (1:5000, ab6721, Abcam) or goat anti-mouse IgG H&L (HRP) (1:5000,ab6789, Abcam) was used to co-incubate with PVDF at 37°C for 1 h. Proteins were detected by ECL, and quantitatively analyzed by scanning densitometry (Bio-Rad, United States).

The processes of fixation, decalcification, embedding, and sectioning of the femoral heads were the same as those described for H&E staining. For immunohistochemical staining, the slices were subjected to deparaffinization; antigen retrieval; blocking in horse serum for 30 min; incubation with primary antibodies against Osterix (dilution 1: 100, Abcam: #ab209484), runt-related transcription factor 2 (RUNX2) (dilution 1: 100, Abcam: #ab236639), and osteocalcin (OCN) (dilution 1: 100, Abcam: #ab93876) for 12 h; and then incubation with the appropriate biotinylated secondary antibodies. All antibodies were purchased from Abcam (Cambridge, UK). Finally, the slices were stained with diaminobenzidine and counterstained using hematoxylin. Section images were acquired using an Axiovert 5 × and 20 × optical microscope (Zeiss, Germany), and the number of positive cells was used as a quantitative indicator.

For total protein extraction, cells were lysed in RIPA buffer (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1% Protease inhibitor on ice. Protein concentration was determined by the BCA assay (Sigma-Aldrich, St Louis, MO, USA). Next, proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.2 μm polyvinylidene difluoride membrane. The membranes were blocked with 5% skimmed milk and incubated with the primary antibodies anti-RUNX2 (Abcam, Cambridge, MA, USA ab236639) and anti-OSTERIX (Abcam, ab209484) overnight at 4°C. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, Abcam, ab8245) was used as an internal reference. Next day, secondary antibodies against the species of primary antibodies were added and incubated for 1 hour at room temperature. Signals on the membrane were detected by electrochemiluminescence (ECL, Pierce, Rockford, IL, USA), and the protein band density of immune complexes was analyzed using AlphaEaseFC (Alpha Innotech Corporation, San Leandro, CA, USA) software.