Anti osx antibody

Western blot analysis was performed as previously described [21 (link)]. Macrophages or cementoblasts were washed and solubilized with RIPA lysis buffer. Protein concentrations were measured and adjusted to be the same, followed by electrophoresis on a precast gel and then transferred to a polyvinylidene difluoride membrane. After being blocked with 5% BSA, the transferred membranes were incubated at 4°C overnight with anti-iNOS antibody, anti-ALIX antibody, anti-GM130 antibody (Proteintech, Chicago, IL, USA), anti-CD63 antibody, anti-Arg-1 antibody, anti-BSP antibody, anti-COL-1 antibody (Cell Signaling Technology, Danvers, MA, USA), anti-RUNX2 antibody, anti-OSX antibody, or anti-β-actin antibody (Abcam, Cambridge, MA, USA) diluted at 1 : 1000. Subsequently, the membranes were incubated with anti-rabbit or anti-mouse secondary antibodies (ZB-2301 and ZB-2305, Zhongshan Golden Bridge Biotechnology, Beijing, China), which were diluted at 1 : 5000 at room temperature for 1 h. The expression of associated proteins was visualized with a chemiluminescence reagent and analyzed using ImageJ software.

Cells were rinsed with PBS, trypsinized and collected by centrifugation at 4200 rpm and 4 °C for 5 min. Cell lysis was obtained by incubating in lysis containing RIPA, 1 mM PMSF and complete EDTA-free protease inhibitor cocktail (Roche, Mannheim, Germany). Equivalent amounts of protein (20–40 μg) were subjected to SDS-PAGE (Beyotime, Shanghai, China) and transferred onto a PVDF membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% non-fat dried milk in TBST for 1 h and incubated with specific antibodies at 4 °C overnight. Proteins were detected using HRP-conjugated secondary antibodies and visualized using an enhanced chemiluminescence kit (Millipore, Bedford, MA, USA). The intensity of each band was quantified using Quantity One software (Bio-Rad, Hercules, CA, USA) after normalization to GAPDH. For immunoblotting, anti-PTHR1 antibody (Abcam, Cambridge, UK), anti-Runx2 antibody (Abcam, Cambridge, UK), anti-Osx antibody (Abcam, Cambridge, UK), anti-COL1 antibody (Abcam, Cambridge, UK), anti-OPN antibody (Abcam, Cambridge, UK), anti-ALP antibody (Biorbyt, Cambridge, UK), anti-OCN antibody (Biorbyt, Cambridge, UK), and anti-BSP antibody (Biorbyt, Cambridge, UK) were used at 1:1000 dilution.

The following primary antibodies were used for immunoblotting: anti-Osx antibody (Abcam); anti-Flag antibody (Sigma Chemical Co., St Louis, MO, USA); anti-MMP13, anti-VEGF, anti-CD34, and anti-PTHrP antibodies (Protech, Nanking Dist, Taipei, Taiwan); and anti-β-actin, anti-rabbit, anti-rat, and anti-mouse IgG antibodies (Bioworld Technology, Minneapolis, MN, USA).