Protein Expression Analysis in Cells

The cells of each group were lysed in lysis buffer. Determine the quality of the harvested protein by used BCA kit. Then, 20 μg of total proteins was separated in SDS-PAGE gels and transferred to PVDF membrane. The membranes were blocked for 1 h at room temperature and incubated overnight at 4 °C with the relevant antibodies: anti-ALP antibody (1:1000, Abcam, USA), anti-OCN antibody (1:1000, Abcam, USA), anti-RUNX2 antibody (1:1000, Abcam, USA), anti-SLIT2 antibody (1:1000, Abcam, USA), anti-CD63 antibody (1:1000, Abcam, USA), anti-TSG101 antibody (1:1000, Abcam, USA), anti-HSP70 antibody (1:1000, Abcam, USA) and anti-GAPDH antibody (1:10,000, Abcam, USA). Membranes were rinsed and incubated for 1 h with secondary antibodies (Abcam, USA). After three times of washing, membranes were exposed with the ECL kit. Bands were analyzed using ImageJ software (version 1.6 NIH) to analyze the relative expression levels of the above markers.