Bone tissues
were placed in a mortar, and liquid nitrogen was added, allowing the
callus to be ground to fine particles with a pestle. Subsequently,
the tissue was transferred to a 1.5 mL EP tube for protein isolation.
Total protein was extracted from cells and tissues by RIPA (Beyotime,
P0013B, Shanghai, China) with 1% protease inhibitor cocktail for general
use, 100× (Beyotime, P1005, Shanghai, China). Protein (20 μg)
was separated on a denatured sodium dodecyl sulfate-polyacrylamide
gel and transferred to a polyvinylidene fluoride (PVDF) membrane.
The PVDF membrane was blocked with defat mike (5% dissolved in TBS-T)
for 2 h and then incubated with rabbit polyclonal ALP (1:1000, Abcam),
Runx2 (1:1000, Abclonal), OCN (1:500, Abcam), collagen I (Col1) (1:3000,
Abcam), GAPDH (1:1000, Abclonal), SSRP1 (1; 1000, Proteintech), and
β-catenin (1:1000, Abclonal) for 16 h at 4 °C. After being
washed three times, the membranes were followed by incubation with
horseradish peroxidase-coupled goat antirabbit IgG H&L for 1 h
at 37 °C. The blotting membrane was treated with an ECL chemiluminescent
substrate (Biosharp, BL520A, Shanghai, China) and visualized using
a ChemiDoc MP chemiluminescence gel imaging system (Bio-Rad,1708280,
California, USA).
were placed in a mortar, and liquid nitrogen was added, allowing the
callus to be ground to fine particles with a pestle. Subsequently,
the tissue was transferred to a 1.5 mL EP tube for protein isolation.
Total protein was extracted from cells and tissues by RIPA (Beyotime,
P0013B, Shanghai, China) with 1% protease inhibitor cocktail for general
use, 100× (Beyotime, P1005, Shanghai, China). Protein (20 μg)
was separated on a denatured sodium dodecyl sulfate-polyacrylamide
gel and transferred to a polyvinylidene fluoride (PVDF) membrane.
The PVDF membrane was blocked with defat mike (5% dissolved in TBS-T)
for 2 h and then incubated with rabbit polyclonal ALP (1:1000, Abcam),
Runx2 (1:1000, Abclonal), OCN (1:500, Abcam), collagen I (Col1) (1:3000,
Abcam), GAPDH (1:1000, Abclonal), SSRP1 (1; 1000, Proteintech), and
β-catenin (1:1000, Abclonal) for 16 h at 4 °C. After being
washed three times, the membranes were followed by incubation with
horseradish peroxidase-coupled goat antirabbit IgG H&L for 1 h
at 37 °C. The blotting membrane was treated with an ECL chemiluminescent
substrate (Biosharp, BL520A, Shanghai, China) and visualized using
a ChemiDoc MP chemiluminescence gel imaging system (Bio-Rad,1708280,
California, USA).