The cell total protein analysis process was performed as described in our previous study [20 (link)]. Briefly, the separated proteins were transferred onto polyvinylidene difluoride membranes, then the membranes were blocked with 5% nonfat milk and incubated with primary antibodies and second antibodies in turn. The following primary antibodies were used: rabbit anti-Wnt1(1:1000, Abcam), rabbit anti-β-catenin (1:1000, Abcam), rabbit anti-dentin sialophosphoprotein (DSPP) (1:1000, Abcam), rabbit anti-dentin matrix protein-1 (DMP-1) (1:1000, Abcam), and mouse anti-β-actin (1:1000, Santa Cruz). The second antibodies were goat-anti-rabbit or goat-anti-mouse horseradish peroxidase-conjugated IgG (1:1500, Abcam).
A Nuclear Protein Extraction Kit (Active Motif, Carlsbad, CA, USA) was used to extract the nuclear fractions according to the manufacturer’s instructions. The PVDF membranes were probed with specific antibodies: rabbit anti-β-catenin (1:1000, Abcam) and mouse anti-Lamin B1 (1:2000, Abcam) overnight at 4 °C. Then, they are followed by incubation with the second antibodies: goat-anti-rabbit or goat-anti-mouse horseradish peroxidase-conjugated IgG (1:1500, Abcam).
A Nuclear Protein Extraction Kit (Active Motif, Carlsbad, CA, USA) was used to extract the nuclear fractions according to the manufacturer’s instructions. The PVDF membranes were probed with specific antibodies: rabbit anti-β-catenin (1:1000, Abcam) and mouse anti-Lamin B1 (1:2000, Abcam) overnight at 4 °C. Then, they are followed by incubation with the second antibodies: goat-anti-rabbit or goat-anti-mouse horseradish peroxidase-conjugated IgG (1:1500, Abcam).