Anti β catenin antibody

Cell extracts were obtained by lysis with RIPA buffer (0.5% sodium deoxycholate, 50 mM Tris-HCl; pH 8, 150 mM NaCl, 1% NP40, 0.1% SDS) supplemented with a protease inhibitor cocktail (Roche). Equal amounts of protein (10–50 μg/lane) were loaded into SDS-PAGE gels. After transfer onto nitrocellulose membrane, blots were incubated overnight at 4 °C with the following antibodies: anti-OPN antibody (ab8448 Abcam, Cambridge, UK), anti-β-catenin antibody (1/1000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), anti-SNAI2 (Novus Biologicals, Englewood, CO, USA) anti-actin-HRP antibody (1/2000, Santa Cruz Biotechnology, Inc., Dallas, TX, USA), and anti-CDH1 antibody (1/1000, Santa Cruz, Inc., Dallas, TX, USA), followed by incubation with a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit or anti-mouse at 1/2000 dilution, Cell Signaling, Danvers, MA, USA). Results were revealed with a chemiluminescence ECL detection system (Bio-Rad, Hercules, CA, USA) and visualized on Chemidoc systems (Bio-Rad, Hercules, CA, USA). Protein expression was quantified by densitometric analysis of the immunoblots using Image Lab software v.5.2.1 developed by Bio-Rad, Hercules, CA, USA.

hBMSCs were obtained from Cyagen Bioscience (Guangzhou, China). PD (purity ≥ 94%) was purchased from the National Institutes for Food and Drug Control (Beijing, China). Recombinant human Dickkopf-related protein 1 (DKK1) and Noggin were obtained from PeproTech (Rocky Hill, NJ, USA). Ficoll medium to generate a Ficoll density gradient was purchased from GE Healthcare (Silverwater, Australia). Fetal bovine serum (FBS), low-glucose Dulbecco minimum essential medium (LG-DMEM), and penicillin-streptomycin were obtained from Gibco-BRL (Gaithersburg, MD, USA). An MTT assay kit, β-glycerophosphate, dexamethasone, dimethyl sulfoxide (DMSO), and l-ascorbic acid-2-phosphate were all purchased from Sigma (Steinheim, Germany). Alizarin red was obtained from Aladdin Company, and an alkaline phosphatase activity measurement kit was obtained from Nanjing Jiancheng Company (Nanjing, China). pLent-U6-GFP-Puro vector was obtained from GenePharma Company (China). SYBR® Premix Ex TaqTM II and Prime Script TM RT Master Mix were purchased from Takara Biotechnology Company (Dalian, China). Anti-β-catenin antibody, anti-β-actin antibody, secondary antibodies, and phosphor-β-catenin (p-β-catenin) were obtained from Santa Cruz (Paso Robles, CA, USA). Chemiluminescence reagents were purchased from Pierce (Rockford, IL, USA).