Molecular imaging software version 4

The cells from groups A–D at indicated time points (4, 7, and 14 days) were used for protein extraction. Cells were treated with the lysis buffer (Cell Signaling Technology, Danvers, MA, USA) and protein extracts were dissolved in sample buffer containing 50 mM Tris-HCl, 2% SDS, 10% glycerol, 100 mM dithiothreitol (pH = 6.80). Proteins were separated by SDS-PAGE in 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were decorated with anti-β-catenin antibodies and anti-c-myc antibodies (Santa Cruz Biotechnology, Santa Cruz, CA, USA). Bands were captured and documented through a CCD system (Imagestation 2000 MM, Kodak, Rochester, NY, USA). Blots were stripped and re-probed with anti-tubulin antibodies to demonstrate equal loading and to allow normalization of the protein content. Densitometry of the bands was performed using Molecular Imaging Software Version 4.0 (Kodak).

The cells were treated with the lysis buffer (Cell Signaling Technology), and the protein extracts were dissolved in a sample buffer containing 50 mM Tris-HCl, 2% SDS, 10% glycerol, and 100 mM dithiothreitol (pH = 6.80). Proteins were separated using SDS-PAGE in 10% polyacrylamide gel and transferred to a nitrocellulose membrane. Blots were performed with anti-BMP-2 antibody, anti-VEGF antibody, anti-ALP antibody (Abcam), anti-β-catenin antibodies, anti-GSK-3β antibodies, and anti-C-myc antibodies (Santa Cruz Biotechnology, CA). The bands were captured and documented using a CCD system (Image Station 2000 MM, Kodak, Rochester, NY, USA). The blots were stripped and reprobed with anti-actin antibodies to demonstrate equal loading and to enable between-group protein content normalization. Densitometry of the bands was performed using Molecular Imaging Software Version 4.0 (Kodak).