Western blot analysis
Protein lysates were generated with radio immunoprecipitation assay
lysis buffer. Then protein concentrations were determined using BCA
protein assay kit according to the manufacturer’s protocol. Protein
samples (20 μg/lane) were loaded onto 12% sodium dodecyl
sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) gel and
electrotransferred to a polyvinylidene fluoride (PVDF) membrane. After
blocking with 5% non-fat milk, membranes were incubated overnight at
4˚C with primary antibodies, including rabbit anti-rat Runx2 monoclonal
antibody (1:1,000 dilution), rabbit anti-rat OPN polyclonal antibody
(1:1,000 dilution), and rabbit anti-rat Wnt3a polyclonal antibody (1:400
dilution). Then, membranes were incubated with horseradish
peroxidase-conjugated goat anti-rabbit secondary antibody (1:10,000
dilution) for 1 h at room temperature. Membranes were visualized by
enhanced chemiluminescence (Amersham Pharmacia Biotech; Little Chalfont,
U.K.). Loading differences were normalized using rabbit anti-rat GAPDH
monoclonal antibody (1:2,000 dilution) or rabbit anti-rat Tubulin
polyclonal antibody (1:1,000 dilution). Protein band densities on
scanned films were quantified using ImageJ 1.48u software (National
Institutes of Health, Bethesda, Md, USA) and compared with the control.
Analysis of mineralization
Following 14 days culture, cells were fixed, then incubated with
alizarin red S as mentioned above. For further evaluation, staining was
dissolved in 100 µM cetylpyridinium chloride (CPC) for 1 h at 37˚C. The
optical density (OD) value of the staining dissolved CPC was measured at
562 nm using the ELISA plate reader. All experiments were repeated for
three times, and each experiment in triplicate.