Western Blotting Analysis of Cellular Proteins

Western blotting was carried out as described previously [68 (link)]. Firstly, the total proteins in cell lysates were harvested, and then they were separated using SDS-PAGE gels. Later, the proteins that had been separated were transferred to polyvinylidene difluoride membranes, which were then blocked in the fat free milk (concentration of 5%). Subsequently, the following antibodies were incubated under 4 °C overnight: anti-RUNX2 (#12556, Cell Signaling Technology, 1:1000), anti-TSG101 (#ab125011, Abcam, 1:1000), anti-CD63 (#ab217345, Abcam, 1:1000), anti-Calnexin (#ab213243, Abcam, 1:1000), anti-UCHL3 (#D25E6, Cell Signaling Technology, 1:1000), anti-SMAD1 (#AP20642c, Abcepta, 1:1000), and anti-β-ACTIN (#4976, Cell Signaling Technology, 1:1000). After incubated with horseradish peroxidase‐conjugated secondary antibodies (Shengxing Biological, Nanjing, China) for 1 h, the specific bands were visualized with the enhanced chemiluminescence (Tanon). The relative density was measured using ImageJ software (V1.8.0, National Institutes of Health, USA).