A11753

After two washes with PBS, the cells were treated with radioimmunoprecipitation buffer (Beyotime) containing 1 mmol L⁻¹ phenylmethyl sulfonyl fluoride. Following a 1 minute ultrasonic shock, the MC3T3-E1 cells were centrifuged at 12 000g and stored at 4 °C for 10 minutes. The cell fragments were then discarded to obtain the total protein. The proteins were loaded onto a 7.5% SDS-polyacrylamide gel for electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was incubated in 5% bovine serum albumin for 2 hours and then with primary antibodies, including ALP (1 : 1000, Abcam, ab154100), RUNX2 (1 : 1000, Abclonal, A11753), β-catenin (1 : 1000, Abcam, ab265591), phosphorylation of β-catenin (1 : 1000, Abcam, ab75777), and GAPDH (1 : 2500, Abcam, ab9485), which were incubated overnight at 4 °C. The membrane was visualized using an enhanced chemiluminescent reagent (Abcam), and GAPDH was used as an internal control. Secondary antibodies (1 : 5000, ab6721, Abcam) were applied for 2 hours in the analysis.