2.21. Western blot analysis

Proteins extracted from osteoblasts and mouse left femur were dissolved in radio‐immunoprecipitation assay buffer containing 50mm Tris‐HCl (pH = 7.4), 150 mmol/L NaCl, 0.1% sodium dodecyl sulphate (SDS), 1% sodium deoxycholate, 1 mmol/L ethylene diamine tetraacetic acid, 1% Triton X‐100 and protease inhibitor. The proteins were extracted at 4°C for 30 minutes. After performed with 10% SDS‐polyacrylamide gel electrophoresis (20 μg/lane), the proteins were transferred onto membranes (Millipore, MA, USA), which were blocked with 5% skim milk and incubated with primary antibodies Elf3 (1:1,000, Abcam Inc.) and GAPDH (1:2000) at 4°C overnight. Next, the membranes were incubated with horseradish peroxidase–conjugated secondary antibody (1:2000, Santa Cruz Biotechnology, Inc.). GAPDH was used as the loading control, and proteins were detected using enhanced chemiluminescent kits (Applygen Technologies). The Image‐Pro Plus 6.0 was used to analyse the protein bands.