Western blot analysis
Protein concentrations of cell lysates were determined using a protein
assay kit (Bio-Rad Laboratories, USA). Total proteins (20 μg) were
separated by SDS-PAGE and transferred onto a polyvinylidene difluoride
membrane (Millipore, USA). After blocking in 5% non-fat milk, the
membranes were probed with primary antibodies (RUNX2: sc-101145, GAPDH:
sc-47724, Santa Cruz Biotechnology, USA) overnight at 4 °C, then washed
3 times with TBS-Tween 20 followed by incubation at room temperature 1 h
with a horseradish peroxidase (HRP)-conjugated secondary antibody. The
protein bands were visualized with an Immobilon Western Chemiluminescent
HRP Substrate (Millipore, USA).