Total gsk 3β

Wst-1 assay kit was purchased from Daeillab (Daeillab, Korea). LY294002 (PI3K/Akt inhibitor) and PD98059 (Erk inhibitor), L-Ascorbic acid were purchased from Sigma Aldrich (Sigma Aldrich, USA). BIO (GSK-3β inhibitor) was purchased from Santa Cruz Biotechnology (Santa Cruz Biotechnology, USA). Specific antibodies such as p-Akt, total-Akt, β-actin, Cyclin E, p-cdk2, p21 were obtained from Cell Signaling Technology (Beverly, USA). and p-GSK-3β, total-GSK-3β, p-Erk, total-Erk, Active-caspase-3 antibodies were purchased from Abcam (Cambridge, USA). Muse™ Muse™ Cell Cycle Kit (MCH100106) and Muse™ Cell Analyzer (PB4455ENEU) were purchased from Millipore (EMD Millipore Corporation, Germany). 3M™ Tegaderm (sterile barrier to external contaminants) was purchased from 3 M (3 M, USA).

Proteins in BMSCs were obtained by lysing cells in radio immunoprecipitation assay (RIPA; Beyotime). Bicinchoninic Acid Kit for protein (BCA kit; Sigma-Aldrich) was used to quantify protein concentration. After protein was separated and transmitted to a polyvinylidene fluoride membrane (Bio-Rad Laboratories), membrane was blocked (Beyotime) and incubated with primary antibodies overnight at 4 °C, including CB2 (1:500), Runx2 (1:500), OCN (1:500), Osterix (1:500), pSer9-GSK-3β (1:500), total GSK-3β (1:2000), β-catenin (1:2000), and Axin2 (1:1000, all obtained from Abcam). After incubation with corresponding secondary antibody, the protein bands were captured by enhanced chemiluminescence (ECL; Sigma-Aldrich). Relative gray level was measured for quantitative analysis using Image Lab 3.0.

MC3T3-E1 cells were grown in a differentiation-inducing medium for 3 days, washed, and then collected with 0.5% trypsin (GIBCO, Grand Island, U.S.). Cells were lysed in radio immunoprecipitation assay (RIPA; Beyotime) buffer for 30 min at 4°C and then the supernatant was collected by centrifugation at 14,800 rpm at 4°C. The supernatant was purified, and total protein concentration quantified using a bicinchoninic acid protein kit (BCA kit; Beyotime). Twenty µg total protein were separated using SDS-PAGE (New Cell & Molecular Biotech Co., Ltd., Suzhou, China) and then transferred to a nitrocellulose filter membrane. After blocking with QuickBlock™ blocking buffer (Beyotime), the membranes were incubated with the appropriate primary antibodies for 12 h at 4°C. Antibodies against Runx2 (1:1000), OCN (1:500), Osterix (1:1000), total GSK-3β (1:2000), pSer9-GSK-3β (1:500), β-catenin (1:5000), and Axin-2 (1:1000) were purchased from Abcam. The membranes were rinsed 3 times with TBS-Tween and then incubated at room temperature with secondary antibody (1:5000) for 1 h. Enhanced chemiluminescence (ECL; Sigma-Aldrich, St. Louis, MO, USA) was used to visualize protein bands, and the relative grey level was analyzed using Image J.