2.5. Bone Formation-Related Markers Content by Western Blotting Analysis
SECONDARY, LITTLE
Proteins of the differentiated SaOS-2 cells were extracted with a RIPA buffer (Tech & Innovation, Gangwon, Republic of Korea) containing protease inhibitors (Roche, Basel, Swiss) and separated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated proteins were transferred onto a membrane (Bio-Rad, Hercules, CA, USA) and incubated with mouse and rabbit monoclonal anti-OCN, -RUNX family transcription factor 2 (Runx2), -Osterix and -ALP (Cell Signaling Technology; Danvers, MA, USA). After incubation with anti-mouse and rabbit IgG secondary antibody conjugated with horseradish peroxidase (Cell Signaling Technology), the immunoreactive bands were visualized by a chemiluminescence imaging system (GE Healthcare, Little Chalfont, UK) using enhanced chemiluminescence reagents. The band intensity was quantified using the Image J software (v1.4.3.x., U.S. National Institutes of Health, Bethesda, MD, USA). All experiments were performed in triplicate.