Anti runx2

Cell lysates were extracted in 0.1 M NaCl, 0.01 M tris–HCl (pH 7.6), 1 mM EDTA (pH 8.0), 1 mg/ml aprotinin, and 100 mg/ml PMSF; protein concentrations were determined by a Bio-Rad protein assay. Protein (50 μg) was boiled at 95 °C in sodium dodecyl sulphate (SDS) sample buffer for 5 min, electrophoresed on 10% or 12% SDS-PAGE gels, and transferred to polyvinyldifluoridine membranes. Then, blots were incubated overnight at 4 °C with anti-HOXA9 (Abcam, Cambridge, UK, #ab191178; 1:1000), anti-RUNX2 (Abcam, Cambridge, UK, #ab23981; 1:1000), or anti-β-actin (Bethyl, Montgomery, Texas, USA, #A300-491A; 1:8000) antibodies. Membranes incubated with anti-goat or anti-rabbit secondary antibody (Santa Cruz Biotechnology; 1:5000) at room temperature for 60 min. Protein band signals were visualized using an ECF western blotting kit (Amersham Biosciences, Piscataway, NJ, USA) and detected with an LAS3000 luminescent image analyzer (Fuji Photo Film).

Total protein was isolated using RIPA lysis buffer (Applygen Technologies Inc). BCA assay (Thermo Fisher Scientific, Inc) was used to determine the protein levels. The protein lysate was then separated through SDS‐PAGE (Beijing Biosynthesis Biotechnology) and transferred onto PDVF membrane. After blocking with skim milk (Solarbio Inc) for 1 hours at room temperature, the membrane was added with anti‐Smad3 (1:2000 dilution; Abcam), anti‐RUNX2 (1:2000 dilution; Abcam), anti‐collagen type X (1:2000 dilution; Abcam), anti‐Indian Hedgehog (1:2000 dilution; Abcam), anti‐OPN (1:2000 dilution; Abcam) and anti‐OCN (1:2000 dilution; Abcam) primary antibodies. After overnight incubation at 4°C, the membrane was rinsed in 1 × TBST (Solarbio Inc) and then incubated with HRP‐labelled rabbit antimouse (1:3000 dilution; Abcam, USA) secondary antibody at room temperature for 1 hours. We employed GAPDH as the internal control (1:3000, Abcam). Additionally, when > 1 protein was presented, the Western blotting membranes were stripped and reprobed with WB Stripping buffer (No: 21059; Thermo Fisher Scientific, USA) and different primary antibodies, respectively. The grey values were measured by Image Lab (v 5.2.1).

For the western blot analysis, cells were lysed on ice for 30 min in a lysis buffer containing 50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% Nonidet P-40 and 0.1% SDS supplemented with protease inhibitors (10 mg/ml leupeptin, 10 mg/ml pepstatin A and 10 mg/ml aprotinin). Protein fractions were collected by centrifugation at 15 000 g at 4 °C for 10 min and then subjected to 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA and incubated with specific antibodies overnight at 4 °C. A horseradish peroxidase-labeled secondary antibody was added and visualized using an enhanced chemiluminescence detection system (Millipore, Billerica, MA, USA) as recommended by the manufacturer. We used the following primary antibodies to determine the concentrations of proteins in the lysates: anti-Runx2, anti-Ocn, anti-Col1a1 mAb (1:1000, Abcam, Cambridge, UK), anti-Kdm5a, anti-GAPDH mAb, anti-Histone H3, anti-H3K4me3, anti-H3K9me3, anti-H3K27me3 and anti-smad1/5/8 mAb (1 : 1000, Cell Signaling Technology, Inc., Danvers, MA, USA).

Sildenafil was a kind gift from Pfizer (New York, NY), whereas 8-bromo-cGMP was purchased from Calbiochem. Primary antibodies, anti-Ki67, anti-von Willebrand factor, anti-Osteopontin and anti-Runx2 were purchased from Abcam. The HRP-conjugated secondary antibodies and HRP-detection kit were obtained from Biogenex.

The right femur tissues were homogenized in RIPA buffer using a homogenizer and the homogenate was centrifuged at 12,000 g for 15 min at 4°C. Total proteins of the supernatant were measured using the Bicinchoninic acid Protein Assay Kit (Thermo Fisher Scientific). Protein samples were separated by 15% SDS-polyacrylamide gels, and then transferred onto a PVDF membrane (Roche, USA). After blocking with 5% skim milk for 1 h at room temperature, the membrane was hybridized with the following primary antibodies overnight at 4°C: anti-OPG (1:1,000; Abcam, USA), anti-RANKL (1:1,000; Abcam), anti-RunX2 (1:1,000; Abcam), anti-OCN (1:1,000; Abcam), and GAPDH (1:1,000; Abcam). The membrane was washed with TBST and subsequently incubated with horseradish peroxidase-labeled secondary antibody for 45 min at room temperature. Visualization was performed with chemiluminescence detection reagent (ECL, USA). The relative protein levels were analyzed with ImageJ image analysis software (National Institutes of Health, USA) and normalized to GAPDH.

Total protein was extracted from the cells by lysis in RIPA buffer (10 mM Tris-HCl, 1 mM EDTA, 1% sodium dodecyl sulfate, 1% Nonidet P-40, 1∶100 proteinase inhibitor cocktail, 50 mM b-glycerophosphate and 50 mM sodium fluoride). The protein concentration in the extracted lysates was determined using a protein assay solution based on the absorbance at 595 nm (Bio-Rad, Hercules, CA, USA). Additionally, 20 to 50 mg of the cell lysate samples were separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane (Bio-Rad, Hercules, CA, USA). The membranes were blocked with 5% milk for 2 hours and then incubated with anti-Runx2 (Abcam, Cambridge, UK) and anti-β-actin (Cell Signaling Technology, Beverly, MA, USA) primary antibodies. The immune complexes were then incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse IgG antibodies (Boshide, Beijing, China). Immunodetection was performed using the Western-Light Chemiluminescent Detection System (Peiqing, Shanghai, China).

Immunofluorescence staining was performed as described in previous reports [32 (link), 33 (link)]. Briefly, the cells were grown on coverslips and treated for 3 days. After washing with PBS, the cells were fixed with 4% paraformaldehyde, permeabilized with PBS containing 0.1% (v/v) Triton X-100 (Solarbio), and incubated with 5% bovine serum albumin (Solarbio). After that, the cells were incubated with anti-TRPM7 (rabbit polyclonal antibody, 1:80, GeneTex, Irvine, CA) or anti-RUNX2 (rabbit monoclonal antibody, 1:500, Abcam) primary antibody at 4 °C overnight, followed by incubating with secondary antibody at room temperature for 1 h. The secondary antibody was DyLight 549 goat anti-rabbit IgG (1:200, Abbkine, Wuhan, China) or CoraLite488-conjugated goat anti-rabbit IgG (1:100, Proteintech, Rosemont, IL). If the cells needed microfilaments staining, they were incubated with Actin-Tracker Green (Beyotime) according to the manufacturer’s instructions. The cells were then counterstained with DAPI for 5 min and mounted with an anti-fluorescence quenching agent (Solarbio). The stained cells were observed under a laser scanning confocal microscope. The ImageJ 1.4.3.67, GraphPad Prism 7.0, and Origin 2019 (OriginLab Corporation, Northampton, MA) software packages were used for image processing.