Anti cd63 antibody

To confirm the isolation of exosome, proteins were separated by SDS-PAGE. After the exosome proteins were transferred onto a nitrocellulose membrane (Schleicher and Schuell, Keene, NH, USA), the membrane was blocked with 5% (w/v) non-fat dry milk in Tris-buffered saline (TBS)–0.1% Tween-20 (TBST) at 4 °C. Then, the membrane was incubated with an anti-HSP90 and an anti-CD63 antibody (Cell Signaling) for 5 h. The membrane was incubated with an anti-rabbit secondary antibody coupled to peroxidase (Cell signaling). After a 2 h-incubation at room temperature, the membrane was washed with TBST, and blots were detected with an enhanced chemiluminescence solution (ECL1; Amersham Biosciences).

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.