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Cellular proteins (10–20 μg in 25 mM Tris-HCl pH 8, 0.5% SDS, 0.05% 2-mercaptoethanol, 2.5% glycerol, and 0.001% bromophenol blue) were denatured at 100 °C for 5 min, subjected to SDS-PAGE in a 8–12% polyacrylamide gel, and then electroblotted onto a PVDF membrane at 130 V for 1 h
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Detection indicators: bone density (lumbar spine, hip), bone turnover markers (OC, CTX), bone microstructure (number, thickness, and gaps of bone trabeculae), and skeletal mechanical strength