Anti runx2

Western blotting was carried out as described previously.^{45 (link)} Total protein was extracted from the cultured cells using RIPA lysis buffer (Beyotime, Nanjing, China), which was supplemented with a protease inhibitor cocktail according to the manufacturer’s instructions. The protein mixtures were centrifuged to remove cellular debris. The extracted proteins were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Amersham, Little Chalfont, UK). The PVDF membranes were blocked for 2 h at room temperature using 5% non-fat milk (BD Biosciences, San Jose, CA). The membranes were incubated at 4 °C overnight with the following primary antibodies: anti-OCN (1:1 000; Boster Biological Technology, Wuhan, China), anti-BSP (1:1 000; Boster Biological Technology), anti-ALP (1:1000; Proteintech Group, Wuhan, China), anti-RUNX2 (1:1 000; Proteintech Group), and anti-GAPDH (1:1 000; Boster Biological Technology). Subsequently, the membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (ZSGB-Bio) for 1 h at room temperature. The immunoreactive bands were visualized using an ECL chemiluminescence reagent (Solarbio). The relative intensities were analyzed and compared to their respective controls using ImageJ software (NIH, USA).

Cells (5 × 10⁶ per well) from each group were incubated in separate culture bottles. After 48 h of incubation, the cells were harvested, total protein was extracted, and protein concentrations were measured using a BCA kit (Takara Bio Inc., Shiga, Japan). Next, equal amounts (80 µg) of boiled protein were separated by 4–20% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) using GAPDH as the internal loading reference and then transferred to a polyvinylidene fluoride (PVDF) membrane (Schleicher and Schuell, Keene, NH, United States). After blocking in 5% skimmed milk for 2 h at room temperature, the membrane was incubated with primary antibody (anti-BMP-2, anti-Runx2, or anti-GAPDH, diluted 1:2000) (Proteintech Co., Manchester, UK) at 4°C overnight, washed, and then incubated with horseradish peroxidase (HRP)-labeled goat anti-mouse IgG or goat anti-rabbit IgG (1:2,000, Santa Cruz Biotechnology, Inc., Dallas, TX, United States). Band intensities were measured after detection using enhanced chemiluminescence (ECL) reagent (Thermo).