Western blotting was carried out as described previously [20 (link)]. In brief, PMSF-RIPA lysis buffer was added to the cells and the resultant lysate was centrifuged at 12,000 rpm for 5 mins to permit collection of the supernatant. The concentration of each protein was measured by the BCA method and the concentrations were adjusted to separation by SDS-PAGE. For each sample, we loaded 15 μl (50 μg) per well. Following separation, proteins were transferred to the nitrocellulose membrane. Membranes were then incubated with primary antibodies (anti-CD9, CBL162, Sigma-Aldrich; anti-TSG101, SAB2702167, Sigma-Aldrich; anti-GAPDH, G8795, Sigma-Aldrich; anti-PPARγ, MAB3872, Sigma-Aldrich; anti-Col2, CP18, Sigma-Aldrich; anti-Runx2, AV36678, Sigma-Aldrich; anti-Sox9, AV37986, Sigma-Aldrich; and anti-Aggrecan, MABT83, Sigma-Aldrich) at 1 : 1,000 and secondary antibodies at 1 : 5,000 (goat anti-rabbit (ab6721, Abcam) or goat anti-mouse (ab97023, Abcam) horseradish peroxidase- (HRP-) conjugated secondary antibody), and positive binding was visualized using a ChemiDoc™ XRS imaging system (Bio-Rad, Beijing, China). The immunoreactive bands were analyzed with ImageJ software (National Institutes of Health, Bethesda, MD, USA).
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