Ab65024

Briefly, total proteins from MG63 and U2OS cells were isolated using radio-immunoprecipitation assay (RIPA) lysis buffer and protein concentration was quantified using a bicinchoninic acid (BCA) Protein Assay kit. Equal amount of protein samples (30 μg) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes. Nonspecific binding proteins were blocked with 5% nonfat milk, primary antibodies against MMP2 (Abcam, ab181286, 1:1000), MMP9 (Abcam, ab228402, 1:1000), Collagen 1 (Abcam, ab260043, 1:1000), osteopontin (Abcam, ab214050, 1:1000), RANKL (Abcam, ab65024, 1:1000), Runx2 (Abcam, ab23981, 1:1000), osteocalcin (Abcam, ab181286, 1:5000), DKK1 (Abcam, ab93017, 1:1000), β-catenin (Abcam, ab224803, 1:1000), PTHR1 (Abcam, ab189924, 1:1000), and GAPDH (Abcam, ab181602, 1:1000) were applied to incubate membranes overnight at 4°C. On the next day, secondary antibodies (Abcam, ab205718, 1:50,000) were employed to incubate membranes for 1.5 h at room temperature. Protein bands were developed with electrochemiluminescence (ECL) detection kit (Beyotime, Shanghai, China). Protein expression was analyzed using Image J software with GAPDH as the internal reference.

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.

The expression of RANKL on the osteoblast surface was detected by immunofluorescence technique. Osteoblasts were fixed with 4% paraformaldehyde for 30 min, washed three times with PBS, blocked with 5% bovine serum albumin for 1 h, and then incubated with RANKL antibody (Abcam, ab65024, 1:500) overnight at 4°C. After washing, fluorescent goat anti-rabbit secondary antibody IgG (Abcam, ab150077, 1:500) was added for incubation in the dark for 2 h. After washing, anti-fluorescence quenching sealing agent DAPI was added to seal the membrane, and images were observed and photographed under a fluorescence microscope.