Mouse active β catenin

Cells were harvested in RIPA lysis buffer (Beyotime Co.). Whole-cell protein extracts were quantified by the BCA assay, separated on 12% SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA) and blocked with 5% nonfat milk powder in PBST for 3 h. The membranes were probed overnight with the primary antibodies and then 4 h with peroxidase-conjugated secondary antibodies (Boster, Wuhan, China). Antibodies used in this study included the mouse β-Actin, rabbit Smad1, rabbit phospho-Smad1, mouse GSK3β, rabbit phospho-GSK3β (Cell Signaling, Beverly, MA, USA), rabbit β-catenin (Abcam, Cambridge, MA, USA) and mouse active β-catenin (Millipore). The blots were visualized using an ECL Kit (Amersham Biosciences, Piscataway, NJ, USA) according to the manufacturer's recommended instructions. To measure the protein abundance, gray value of the blots in scanned images was measured with ImageJ Plus software (National Institutes of Health, Bethesda, MD, USA). Gray value of each target protein was normalized to that of β-Actin before comparison.

Decalcified MSC implants were dehydrated in 30% sucrose solutions until submerged in bottom. Samples were embedded into optimal cutting temperature compound (OCT) for frozen sections, and analyzed by immunofluorescence assay following the standard protocol. Antibodies used in this study including goat Smad1 (Santa Cruz Biotechnology), mouse active β-catenin (Millipore), rabbit phospho-Smad1 and rabbit GSK3β (Cell Signaling). The samples were treated with fluorescence-labeled secondary antibodies and the nuclei were stained with 100 ng/ml of 4',6-diamidno-2-phenylinde (DAPI) (Beyotime Co.). All samples were examined under a confocal microscope (Olympus Optical, Tokyo, Japan).