Western blot analysis

Five samples from each group were examined to determine protein levels by western blot analysis. In brief, the subchondral bone specimens were thawed, washed with pre-chilled distilled water (to remove bone marrow cavity contents), weighed and homogenized in liquid nitrogen. Total protein was extracted using the total protein sample kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions, and the protein concentration was determined using the bicinchoninic acid (BCA) method. Subsequently, proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto a polyvinylidene fluoride membrane at 300 mA for 90 min. After 3 washes in TBST (5 min each time), the membrane was blocked in Tris-buffered saline-Tween-20 (TBST) containing 5% non-fat milk for 1 h, and treated with rabbit anti-human monoclonal antibodies raised against β-catenin (1:500), TCF-4 (1:1,000; ab185736), sclerostin (1:300), or β-actin (1:2,000; ab8227) (all from Abcam) in TBST at 4°C overnight. After 3 washes in TBST, the membrane was incubated with HRP-conjugated goat anti-rabbit IgG (1:2,000; Beijing Zhongshan Golden Bridge Biotechnology, Co., Ltd.) at room temperature for 1 h. Visualization was carried out by electrochemiluminescence, with the protein bands revealed on an ALS4000 gel image analysis system (GE Healthcare Life Sciences, Logan, UT, USA). Quantity One software (Bio-Rad Laboratories, Inc.) was employed to assess the protein bands, with target protein expression normalized to β-actin levels.