Total protein extraction reagent kit

Total cell protein was extracted using the Total Protein Extraction Reagent Kit (KeyGEN, Jiangsu, China). Protein concentrations were monitored using a BCA Protein Assay Kit (KeyGEN). A total of 30 µg protein was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred onto a polyvinylidene difluoride membrane (EMD Millipore, Billerica, MA, USA). The membranes were cut into pieces according to the molecular weight of each protein after being blocked with 5% non-fat milk. Then, the membranes were incubated with primary antibodies against GAPDH (rabbit monoclonal), β-catenin, c-Myc, or cyclin D1 (1:2000, Abcam, Cambridge, UK) overnight at 4°C. The membranes were then incubated with a secondary antibody (1:5000, Biogot Technology, Nanjing, China) for 2 h at room temperature. An enhanced chemiluminescence substrate (EMD Millipore) was used to visualize the protein bands.

Tissue proteins of the frozen clinical human samples and total cellular proteins of the BMDMs, MC3T3-E1 cells, and RAW264.7 cells were extracted with a total protein extraction reagent kit (KeyGEN, Nanjing, China), following the manufacturer's protocol. Exactly 30 μg protein was loaded per well, subjected to 10% SDS-PAGE, and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were incubated with non-fat milk for 1 h, then with primary antibodies, including anti-p-STAT3 (1:1000, HuaBio, #ET1603-40), anti-STAT3 (1:1000, HuaBio, #ET1607-38), anti-β-actin (1:1000, Cell Signaling Technology, #4970, Danvers, MA, USA), anti-Runx2 (1:1000, Abcam, #ab264077), anti-Osterix (1:1000, Abcam, #ab22552), and anti-NFAT2 (1:1000, Abcam, #ab25916), overnight at 4°C, followed by incubation with goat anti-rabbit IgG HRP-conjugated secondary antibody (ZSGB-BIO, Beijing, China) for 1 h at room temperature. Relevant protein/antibody bands were detected with an enhanced chemiluminescence detection kit (Epizyme, Shanghai, China) and exposed in a Molecular Imager (Bio-Rad, Hercules, CA, USA).