Anti aggrecan

Western blotting was carried out as previously described [10 (link)]. The cells were harvested by RIPA and endogenous proteins were isolated from cell lysates, then the protein concentration was measured by BCA protein assay kit. Equivalent amounts of proteins (20,000 ug) were separated on a 10% SDS-PAGE gel and transferred to 0.22 μm polyvinylidene difluoride membranes. The membranes were blocked by 5% fat-free milk and then incubated with primary antibodies overnight at 4°C. After being washed by TBST for 5 min, the membranes were incubated with secondary antibodies for 1 h. The protein bands were visualized with ImmunoStar Western C (LI-COR, USA). The primary antibodies were listed as follows: anti-Akt(CST, USA, 9272, 1:1000), anti-phospho-Akt(CST,USA,9271, 1:1000), anti-PTEN(CST,USA,9559, 1:1000), anti-GAPDH(CST, USA, 2118, 1:1000), anti-β-actin(CST,USA,3700, 1:1000), anti-collagen II(Servicebio, China, GB11021, 1:2000), anti-aggrecan(Proteintech, USA, 13,880-1-Ap, 1:1000) anti-CD63(SBI, USA, EXOAB-CD63A-1, 1:1000), anti-CD81(SBI, USA, EXOAB-CD81A-1, 1:1000), CD9(SBI, USA, EXOAB-CD9A-1, 1:1000).