Scanning densitometry

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).

Western blotting analysis was used to analyze the expression of proteins. Briefly, cell lysis was performed using radio immunoprecipitation assay lysis buffer containing protease and phosphatase inhibitor cocktails, and the protein concentrations were determined using a BCA assay. Subsequently, proteins (50 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis for separation, after which they were electro-transferred onto a polyvinylidene fluoride membrane. After blocking with 5% BSA for 3 h at room temperature, the membranes were incubated with primary antibodies (CXCL12, Proteintech, 17402-1-AP; β-tubulin, Immunoway, YM3030; GAPDH, Immunoway, YM3029) at 4°C overnight followed by incubation with secondary antibodies (HRP-conjugated Affinipure Goat Anti-Rat IgG (H + L), Proteintech, and SA00001-15) for 2 h. Immunoreactive bands were visualized using a chemiluminescence kit, and the density of the bands was determined using scanning densitometry (Bio-Rad, Hercules, CA, USA).

The preparation of bone marrow cells was the same as above. For western blot analysis, total protein was extracted first, and cells were washed twice with cold PBS and lysed in the radioimmunoprecipitation assay (RIPA) buffer (20 mM Tris-HCl, pH 7.5; 150 mM sodium chloride; 1% Triton; 1 mM EGTA; 1 mM EDTA; 2.5 mM sodium pyrophosphate; 1 mM β-glycerophosphate; 1 mM sodium orthovanadate; 1 μg/ml leupeptin; and 1 mM PMSF) for 20 min on the ice. After centrifugation at 12000×g for 10 min, protein was then quantitated by using a bicinchoninic acid protein assay kit (Bioworld Technology, USA). After being heated at 95°C for 8 min, the samples were loaded on 12% SDS-polyacrylamide gel for electrophoresis and transferred to PVDF membrane (Millipore, Billerica, MA). The membrane was blocked for 2.5 h in TBS-T buffer (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.1% Tween-20) containing 3% (w/v) bovine serum albumin (BSA; Sigma-Aldrich, USA) at room temperature and then probed with each primary antibody (1 : 500 dilution) at 4°C overnight. After washing with TBS-T for three times, the membrane was then incubated with appropriate horseradish peroxidase-labeled secondary antibody for 2 h at room temperature. For quantitative analysis of immunoblot bands, the densities of the bands were measured by scanning densitometry (Bio-Rad, Hercules, CA). All experiments were repeated at least three times.