Western blot analysis

To obtain whole cell protein extracts, low and high passaged undifferentiated and osteo-differentiated cADMSCs and cBMMSCs at d7, d14 and d21 were collected in cell lysis buffer (Boston Bioproducts, Ashland, MA). Whole cell extracts were sonicated and centrifuged to obtain the total proteins. Total protein in each sample was quantitated and concentrations were obtained using the BCA assay (Pierce, Thermo Scientific, Rockford, IL).

For western blot analysis, 25 μg of proteins were electrophoretically separated by 10 % SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The membranes were blocked with 5 % bovine serum albumin and incubated with 1–5 μg of primary antibodies against β-Tubulin (Santa Cruz, Dallas, Texas), osteopontin, (Abcam, Cambridge, MA), BMP-7, (Millipore, Darmstadt, Germany), p38 MAPK (Cell signaling, Danvers, MA), and p44/42 MAPK(Cell Signaling) proteins. Bound proteins were visualized with HRP-conjugated anti-rabbit IgG for β-Tubulin, osteopontin, p38 and p44/42, or anti-mouse IgG for BMP-7. Enhanced Chemiluminescence assay was used for the visualization of the protein bands. The intensity of each band was analyzed using Image J software (Image j 1.48 V, Wayne Rasband, National institutes of health, USA, http://imagej.nih.gov/ij, Java 1.6.0–20 (64-bit).