Ab200369

Protein was extracted by using RIPA buffer (Beyotime, Shanghai, China). 10% SDS-PAGE was used to separate the protein samples. Then, the protein was transferred into PVDF membrane. After blocking with 5% nonfat milk, the protein was incubated with OPG (ab73400, 1 : 1000, Abcam), RANK (ab200369, 1 : 1000, Abcam), RANKL (ab9957, 1 : 1000, Abcam), STAT3 (ab119352, 1 : 1000, Abcam), and GAPDH (ab8245, 1 : 2000, Abcam) primary antibodies at 4°C overnight. Then, peroxidase-conjugated secondary antibody (ab7090, 1 : 2000, Abcam) was added to incubate the protein for 1 h at room temperature. The protein bands were developed by using the ECL system (Pierce, Rockford, IL, USA). The density of the bands was analyzed utilizing the Quantity One software.

Whole-cell extracts were prepared in RIPA buffer containing protease inhibitor cocktail (Roche). The total protein concentration was determined with DC Protein Assay (Bio-Rad Laboratories). Equivalent doses of proteins were then size-fractionated in 10% SDS-PAGE, transferred to a 0.45 μm pore size PVDF Immobilon-P membrane (Millipore), subjected to 5% BSA blockage, and then incubated with the following antibodies: TLR4 (#ab183459, Abcam), RANK (#ab200369, Abcam), TRAF6 (#ab33915, Abcam), and β-actin (#sc-81178, Santa Cruz Biotechnologies. The bound antibodies were detected by the respective horseradish peroxidase-conjugated anti-IgG antibody (Amersham Biosciences) followed by ECL detection system (Amersham Biosciences) according to the manufacturer's instructions.

The frozen comminuted bone tissue specimens were minced and homogenized using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Jiangsu, China). After centrifugation at 5000 rpm at 4°C for 10 min, the supernatant was collected and stored at −80°C. The total protein concentration was determined using a BCA protein assay kit (Thermo, USA). Equal amounts of protein (20 μg) were electrophoresed using 10% SDS-polyacrylamide gel electrophoresis (Merck, Germany) gels and transferred onto a 0.45 μm polyvinylidene fluoride (PVDF) membrane (Millipore, USA). Furthermore, the PVDF membranes were blocked with 5% skim milk in tris-buffered saline (Merck, Germany) for 2 h and then incubated with primary antibodies, including those against secretin (1 : 1000, PA5-75673, Invitrogen, USA), OPG (1 : 1000, ab227387, Abcam, UK), RANK (1 : 1000, ab200369, Abcam, UK), and RANKL (1 : 1000, ab65024, Abcam, UK). The membrane was washed with PBS and incubated with the secondary horseradish peroxidase-conjugated goat anti-mouse/rabbit antibodies (1 : 2000, Abcam, UK) for 0.5 h at room temperature. After incubation, protein bands were quantified using ImageJ 1.52v software.