Cell lysis buffer

Co-immunoprecipitation is a method which can be used to detect the interaction between known proteins using the specificity of antigen and antibody. In the experiment, HEK293T cells transfected with plasmids expressing the FLAG and EGFP tags were harvested at 30 h post transfection with cell lysis buffer (Beyotime). Input samples were prepared from the cell lysate. The remaining lysate was mixed with anti-FLAG M2 magnetic beads (Sigma-Aldrich) and shaken gently on a roller shaker for 1–2 h. Subsequently, the magnetic beads were washed three times with cell lysis buffer and incubated with 2 × SDS-PAGE loading buffer (TaKaRa) for 8 min at 100°C to elute the bound protein. Then, the beads were removed and immunoprecipitated proteins were analyzed using western blotting. The FLAG and EGFP antibodies were used to detect the expression of the target protein via western blotting assay with input samples. The samples incubated with the anti-FLAG M2 magnetic beads were used for detecting whether the two labeled proteins can bind by western blotting with the EGFP antibody.

Exosomes were isolated by Minute™ Hi-Efficiency Exosome Precipitation Reagent (Invent EI-027) according to the protocol and then filtered with a 0.22 μm filter to purify the exosomes. The exosomes were identified by transmission electron microscope observation and exosomal protein biomarkers. The exosomes were fixed with 4% glutaraldehyde at 4° C for 2h, and then rinsed three times with 0.1mol/L PBS and fixed with 1% osmium tetroxide for 2h. Next, dehydrated by conventional ethanol and gradient acetone, exosomes were immersed, embedded, polymerized in epoxy resin and then observed under a transmission electron microscope. The proteins from exosomes were lysed with cell lysis buffer (Takara 635656). CD63 (Abcam 125011) and TSG101 (Abcam 134045) were used as exosome markers.