Sc 73631

Sterilized MBG and BD-MBG samples (20 mg) were co-cultured separately with BMSCs (1×10 4 cells/cm 2 ) for 14 days in osteogenic differentiation medium (n=6). Total protein extracted from cells was fractionated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred electrophorectically onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Subsequently, the membranes were blocked using 5% non-fat milk at room temperature for 1 h. The blots were then incubated overnight with primary antibodies recognizing COLI (1:400, sc-59772, Santa Cruz Biotechnology, Santa Cruz, CA, USA), OPN (1:400, sc-73631, Santa Cruz Biotechnology), OCN (1:400, sc-390877, Santa Cruz Biotechnology), and β-actin (1:1000, 3700, Cell Signaling Technology, Danvers, MA, USA). The blots were then incubated with horseradish peroxidase-conjugated secondary antibody (1:5000, Zhongshan Goldenbridge Biotechnology, Beijing, China). Immunoreactive proteins were visualized using enhanced chemiluminescence. A densitometer (Syngene Bioimaging System; Frederick, MD, USA) and Scion Image software (Frederick) were used to quantify the density of the bands on the blots. The level for β-actin was used to normalize the protein levels for each sample 18) .