Cell protein extraction kit

Protein was isolated from rBMSCs treated with leptin with 10³ ng/ml using a Beyotime Cell Protein Extraction kit (Beyotime Institute of Biotechnology) according to the manufacturer's protocol. The total concentration of protein was determined using the bicinchoninic acid assay method. Proteins (20 µg/ml; 0.6 µg/lane) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes. The membranes were blocked for 30 min with 5% fat-free milk at room temperature and incubated with the following primary antibodies: Anti-ERK1/2 (1:1,000), anti-p-ERK1/2 (1:800), anti-JNK (1:1,500), anti-p-JNK (1:1,000), anti-p38 (1:500), anti-p-p38 (1:800), anti-p^90rsk (1:1,000) and anti-p-p^90rsk (1:1,000) for a minimum of 1 h at 37°C. Membranes were subsequently incubated with horseradish-conjugated (H+L) secondary antibodies (1:4,000) at 37°C and developed using a Pierce™ enhanced chemiluminescence plus western blotting substrate (cat. no. 32132; Thermo Fisher Scientific, Inc.). UVP VisionWorksLS software (UVP, LLC; DBA Analytik Jena US, Upland, CA, USA).

Total proteins were extracted from rMSCs using a Beyotime Cell Protein Extraction kit (Beyotime Institute of Biotechnology, Haimen, China) according to the manufacturer's protocol. Proteins (20 µg) were separated by 12% SDS-PAGE and transferred to nitrocellulose membranes for immunoblotting. Membranes were blocked with 5% fat-free milk for 30 min at room temperature in PBS buffer containing 0.1% Tween-20, and subsequently incubated with primary antibodies against β-actin (1:2,000), ALP (1:800), Runx2 (1:1,000), β-catenin (1:1,500), GSK3β (1:1,000) and phosphorylated-GSK3β (1:8,00) overnight at 4°C. The membranes were subsequently washed three times with PBS, and then incubated with peroxidase-conjugated secondary antibodies IgG (cat no. 9087; 1:4,000; Cell Signaling Technology, Inc.) at 37°C for 1 h. ECL reagents were used to visualize the results (Pierce; Thermo Fisher Scientific, Inc.; cat no. 32132).