Anti smo

The MG63 cells were seeded onto the four different titanium surfaces in 12-well plates with a concentration of 5×10⁴/well, and cyclopamine treatment processes were the same as those in the cell proliferation assay. After 7 days of incubation, the total proteins were extracted from the cells using the RIPA lysis buffer. The concentrations of protein were detected using the BCA Protein Assay Kit. Then, the total proteins (25 μg per lane) were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and, then, transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). After blocking with 5% nonfat milk at 37°C for 2 hours, the membranes were incubated overnight at 4°C with anti-Shh (rabbit monoclonal antibody, 1:500 diluted, ab53281; Abcam, Cambridge, UK), anti-Smo (rabbit polyclonal antibody, 1:800 diluted, sc-13943; Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Gli1 (rabbit polyclonal antibody, 1:500 diluted, #2534; CST, Danvers, MA, USA), and anti-β-actin antibody (1:2,000 diluted; Santa Cruz Biotechnology), followed by horseradish peroxidase-linked secondary antibodies. The immunoreactive bands were visualized by using an enhanced chemiluminescence (ECL) detection kit (Millipore). The quantification of protein was calculated by densitometry analysis using the Quantity One 4.6.6 software.

Cells were homogenized and lysed with RIPA lysis buffer (100 mM NaCl, 50 mM Tris–HCl, pH 7.5, 1% Triton X-100, 1 mM EDTA, 10 mM β-glycerophosphate, 2 mM sodium vanadate, and protease inhibitor), and protein concentrations were measured by using the Bradford method. Equal amounts of protein (30 µg of protein each lane) were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore, Boston, MA, USA). The membrane was blocked for 1 h at room temperature with SuperBlock T20 TBS Blocking Buffer (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membrane was immunoblotted with primary antibodies (anti-PFTK1, anti-E-cadherin, anti-vimentin, anti-Shh, anti-Smo, anti-Ptc, anti-Gli-1, and anti-GAPDH) (Santa Cruz Biotechnology) overnight at 4°C. The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies at 37°C in the blocking buffer for 1 h. The blots were developed using an enhanced chemiluminescence Western blotting detection system (Amersham Bioscience, UK).

On day 63, hind paws were dissected from the CIA mice, fixed in 4% paraformaldehyde for 24 h, and decalcified in 14% EDTA decalcification solution (Servicebio, G1105) for at least 28 days. Decalcified paws were embedded in paraffin blocks and serially sectioned at 4-μm thickness. Paraffin sections were subjected to antigen retrieval using citrate antigen retrieval solution (Servicebio, G1202). The slides were treated with 3% hydrogen peroxide to quench endogenous peroxidase activity, followed by blocking with 5% BSA. The sections were incubated in primary antibodies, including anti-SMO (1:40; Santa Cruz, sc-166685) and anti-GLI1 (1:200; Cell Signaling, 2643) in a moist chamber at 4°C overnight and then incubated in anti-mouse IgG HRP-conjugated secondary antibody (Servicebio, G1214) for 1 h. HRP staining was developed using the DAB (3,3′-diaminobenzidine) Chromogenic Kit (Servicebio, G1212), followed by counterstaining with hematoxylin. The integrated optical density (IOD) value was calculated by assessing the area and density of the dyed region of the immunohistochemistry section using ImageJ software (NIH). The relative IOD was presented as fold change relative to the normal control group.