Tcf 1

Western blot analysis was performed as described previously^{30 (link)}. Cells were lysed in ice-cold RIPA lysis buffer, which was supplemented with protease inhibitor cocktail (Sigma). Nuclear proteins and cytoplasmic proteins were obtained for β-catenin detection using a Cell Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, China). A BCA protein assay kit (Beyotime) was then used to detect the concentration of the protein samples. Thirty micrograms of total protein was separated by 10% SDS-PAGE gels and then transferred to nitrocellulose membranes, followed by 1 h incubation with 5% nonfat milk blocking buffer. The membranes were incubated with primary antibodies against TCF-1 (1:1000 dilution, Abcam), RUNX2 (1:2000 dilution, Abcam), OCN (1:1000 dilution, Abcam), OPN (1:3000 dilution, Abcam), β-catenin (1:500 dilution, Abcam), and GAPDH (1:1000 dilution, Abcam) at 4 °C overnight. The membranes were washed three times in 0.1 M PBST and incubated with HRP-conjugated secondary antibodies (Abcam) for 1 h at room temperature. Bands were developed using chemiluminescence substance (Thermo Fisher Scientific). The proteins were quantified using Quantity One software (Bio-Rad Laboratories, Inc., USA).