Cd9 antibody

The cartilage and chondrocytes were treated with the RIPA lysis buffer (Epizyme, PC101, China) added with a protease inhibitor cocktail (Epizyme, GRF101, China) and a phosphatase inhibitor cocktail (Epizyme, GRF102). The protein contents were calculated using a BCA protein assay kit (Takara, T9300A), and the samples was instantly boiled for 10 min with the addition of loading buffer. An equal quantity of protein extracts (20 μg) was placed onto a sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gel for electrophoresis and transmitted to a PVDF membrane. Following that, the PVDF membrane was sequentially incubated with primary and secondary antibodies. Finally, the enhanced chemiluminescence (BI, 20-500-120) was used to react with secondary antibodies and the images were acquired. The mTOR-antibody (CST, 2983), P-p70S6 (CST, 9234), Calnexin-antibody (Abcam, ab133615, USA), CD9-antibody (Abcam, ab263019), CD81-antibody (Abcam, ab109201), TSG101-antibody (Abcam, ab125011), Alix-antibody (CST, 92880), Lamp2b-antibody (Abcam, ab18529), anti-rabbit IgG, HRP-linked Antibody (CST, 7074) were used as the antibodies.

Purified exosomes were incubated with free CVB3 virion or CVB3-GFP virion at approximately 1:3 ratio of numbers(0.5 mL total volume mixture in PBS). After 4h, Mixture was subjected to CD9 antibody(#ab236630, Abcam) followed by Protein A/G Magnetic Beads (Millipore)with 25μl for 2h. Mixture placed into the magnetic stand to capture the immune complex beads and washed with cold PBS five times. Magnetic beads resuspended in 100μl Release Buffer(20mM trietholamine, 2M NaCl, pH = 6.0) in tubes and incubated for 1h at room temperature while shaking at 800 rpm to release the magnetic beads. Placed the tubes on magnetic stand to collect the supernatant with the released exosomes-CVB3 complex and ready for Nanoparticle-tracking analysis.