Protease inhibitor and a phosphatase inhibitor

To determine the protein expression of osteoclast‐related markers, BMMs (1.2 × 10⁵/well) were seeded into 6‐well plates and cultured with 0 or 1000 ng/mL IGFBP7 for 0, 1 and 3 days. To discover the underlying signalling pathways, RAW264.7 cells (5 × 10⁵ cells per well) were seeded into 6‐well plates. RAW264.7 cells were pre‐treated with vehicle or IGFBP7 (1000 ng/mL) for 6 h and thereafter exposed to RANKL (100 ng/mL) for the indicated time (0, 5, 10, 20, 30 and 60 min). Total cellular protein extracts were obtained using RIPA lysis buffer with a protease inhibitor and a phosphatase inhibitor (Beyotime). Equal amounts of protein extracts were separated by 10% SDS‐PAGE and then electroblotted to PVDF membranes (Millipore). The membranes were thereafter blocked with 5% BSA for 1 hour and then incubated with primary antibodies at 4°C overnight. After that, the membranes were probed with secondary antibodies (BOSTER) for 2 hours at 4°C. The immunoreactive bands were detected by an enhanced chemiluminescent detection reagent (Millipore) in a Bio‐Rad XRS chemiluminescence detection system (Bio‐Rad).

The procedure for Western blotting was as follows: the total protein was first extracted from the cell using a Protease inhibitor and a phosphatase inhibitor (Beyotime Biotechnology) and the concentration of the protein was determined by a BCA assay. The total proteins were extracted from the cells using protease inhibitor and phosphatase inhibitors (Beyotime Biotechnology) and centrifuged for 20 minutes at 12,000 g/min. The supernatant was collected and the concentration of the proteins was determined by BCA assay (Beyotime Biotechnology). Then, after electrophoresis and membrane transfer, the protein on PVDF membrane was incubated with the first antibody of GYS2 (1:500, Sigma), P53 (1:1000, Cell Signaling Technology), P21(1:1000, Cell Signaling Technology), and Cyclin D1(1:1000, Cell Signaling Technology) overnight at 4°C for 1 hour at room temperature and β-Actin (1:5000, cell signaling technology) was used as reference. The membrane was washed 3 times with TBST, and then incubated with secondary antibody at room temperature for 1 hour. The obtained bands used ECL to visualize and quantify using Gel-Pro analyzer.