Enhanced chemiluminescence substrate kit

Cultured MC3T3-E1 cells were harvested in lysis buffer (Beyotime) after different extracts had been supplemented with 100 nM dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerophosphate for 48 h. Protein concentration was measured using a BCA kit (Thermo Scientific). Total protein (80 μg) was separated on 8% polyacrylamide gel and transferred onto polyvinylidene difluoride membranes (Merck Millipore, Billerica, MA, USA). Membranes were blocked for 2 h at room temperature in 5% non-fat powdered milk in Tris-buffer, followed by incubation at 4 °C with primary antibodies to β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), β-catenin, p-β-catenin, Runx2 (Cell Signaling Technology, Danvers, MA, USA), GSK-3β, and p-GSK-3β (Abcam, UK). Bound primary antibodies were recognised by horseradish peroxidase-linked secondary antibodies (Santa Cruz Biotechnology). Protein bands were visualised using an enhanced chemiluminescence substrate kit (Millipore) and exposed to a ChemiDoc™ XRS chemiluminescence imaging system (Bio-Rad Laboratories, Hercules, CA, USA).

Protein was obtained from skeletal muscle tissue or L6 myotubes using radioimmunoprecipitation assay buffer containing a cocktail of protease and phosphatase inhibitors (Thermo Fisher Scientific). Protein concentrations were determined with a BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein (35 μg) from each sample were separated by 10% SDS-PAGE and were subsequently transferred onto polyvinylidene difluoride membranes (Millipore Corporation, Bedford, MA, USA). Antigens were visualized by sequential treatment with specific antibodies, secondary antibodies conjugated to horseradish peroxidase, and an enhanced chemiluminescence substrate kit (Millipore Corporation). The intensities of bands were quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).

OPCs and OLs cells were lysed in a 20 mM Tris and 1% sodium dodecyl sulfate (SDS) pH 7.4 solution pre-heated to 95°C. Using a cell scraper, lysate was collected into microfuge tubes and placed on a 95°C heating block for 10 min. Protein concentration was determined following the manufacturer’s instructions of the Bio-Rad DC Assay (Biorad). Protein lysates were electrophoresed on 10 or 12% SDS polyacrylamide gels (Biorad). After SDS-PAGE, proteins were transferred to a 0.45 μm nitrocellulose membrane (GE Healthcare) using western blotting, and blocked with a 4% bovine serum albumin (Sigma-Aldrich) in 1x tris buffered saline +0.1% tween (Sigma-Aldrich) solution (TBST). After blocking, the membrane was incubated overnight with the following primary antibodies: 1:750 rabbit AMPKα (Cell Signaling, 5,831), 1:750 rabbit phospho-AMPKα (Thr172) (Cell Signaling, 2,531), 1:750 rabbit ACC (Cell Signaling, 3,676), 1:750 rabbit phospho-ACC (Ser79) (Cell Signaling, 11,818), and 1:750 rabbit Histone H3 (Santa Cruz Biotenchnology, FL-136). The next day the primary antibodies were washed 3 times with 1x TBST. Next, membranes were incubated in 1x TBST for 1 h with 1:2000 anti-rabbit horseradish peroxidase linked secondary antibodies (Cell Signaling, 7,074). Enhanced chemiluminescence substrate kit (Millipore) was used to develop protein blots on radiography films (Thermo Scientific).