Protein Extraction and Western Blot Analysis

Total protein extraction was performed using cell lysis buffer (Ambion), and protein quality was determined using a bicinchoninic acid (BCA) protein assay (Pierce, USA). Briefly, 20 μg of protein was denatured at 95°C for 10 min in 5% β-mercaptoethanol solution and 95% Laemmli sample buffer (Bio-Rad). After separation by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Germany) and blocked for 1 h at 25°C using 0.5% skimmed milk. Thereafter, the membrane was incubated with ALP (ab229126; Abcam, USA), RUNX2 (ab236639; Abcam), and GAPDH (ab8245; Abcam) at 25°C for 1 h, followed by secondary antibodies (Bethyl, USA) at 25°C for 1 h. Membrane-bound antibodies were assessed by chemiluminescence after rinsing with PBS [32 (link)].