Detection of exosomal proteins by Western blotting using anti-CD81 antibody

User report

Akiko Takahashi Foundation for Cancer Research Cancer Research Institute Cell Senescence Project Chief Researcher

Products

• Anti CD81, Human (Mouse) Unlabeled, 12C4 (Product number: CAC-SHI-EXO-M03)

Manufacturer: Cosmo Bio Co., Ltd.

■ Exosome monoclonal antibody Anti CD9, CD63, CD81

This product is an antibody that specifically recognizes CD9, CD63, and CD81 known as exosome markers, and is an antibody that can isolate exosomes from serum and culture supernatant using immunoprecipitation method.

• Recognize exosome membrane proteins CD9, CD63, CD81 with high specificity
• Exosome surface antigen protein, endogenous RNA (miRNA), useful for protein analysis

Experiment content

 

Exosomes are extracellular membrane vesicles with a diameter of 50-150 nm that are secreted outside the cell via multivesicular endosomes (multivesicular bodies), in which intraluminal vesicles are formed in late endosomes by the ESCRT complex and ceramide synthase. is. Since exosomes transfer various intracellular molecules such as proteins, lipids, and nucleic acids to other cells, exosomes have recently been reported to function as an intercellular communication tool and have attracted a great deal of attention.

In the process of analyzing the senescence-associated secretory phenotype (SASP), in which senescent cells highly express and secrete inflammatory proteins such as inflammatory cytokines and chemokines, we found that senescent cells are not only responsible for inflammatory proteins. We also found that the secretion of exosomes, a type of extracellular secretory membrane vesicles, was enhanced ¹ . Using normal human fibroblasts, we induced cellular senescence by mitotic lifespan (replicative senescence) and cellular senescence by activated oncogene (RasV12) (oncogene-induced senescence), and removed the exosome fraction in the serum in advance. After culturing in the medium for 48 hours, exosomes in the culture supernatant were collected by ultracentrifugation. Then, when the amount of exosomes secreted by cells was measured by the NTA method (Nanoparticle Tracking Analysis), it was found that the amount of exosomes secreted by senescent cells was about 30 to 50 times higher than that of young cells (Fig. 1, upper row). Furthermore, in order to comparatively analyze the amount of exosome marker proteins secreted by the cells, Western blotting was performed using the collected exosome fractions. At this time, when the anti-human CD81 monoclonal antibody (SHI-EXO-M03) sold by Cosmo Bio Inc. was used at a dilution of 1:1000, senescent cells clearly secreted exosome proteins compared to young cells. An increase was confirmed (Fig. 1, bottom). We have also observed that exosomes, which are highly secreted by senescent cells, promote the growth of breast cancer cells (MCF-7) ² .

Fig.1 Analysis of exosomes secreted by normal human fibroblasts (TIG-3). ^*1
Exosome measurement result by NTA method and detection of exosome marker protein by Western blotting method.

*1
©Akiko Takahashi (Japanese Foundation For Cancer Research) Nature Communications volume 8, Article number: 15287 (2017) ( Licensed under CC BY 4.0 )

Exosomes have attracted particular attention for their function as a communication tool between cells and their usefulness as a diagnostic tool, and research has progressed so far. There are many parts of We would like to clarify the biological significance of exosome secretion by analyzing the detailed molecular mechanism by which normal cells secrete exosomes. Furthermore, by analyzing the functions of exosomes as SASP factors secreted by senescent cells, we would like to elucidate the role exosomes play in vivo and the relationship between exosomes and age-related diseases such as cancer.

References

1. Takahashi, A. et al . Exosomes maintain cellular homeostasis by excreting harmful DNA from cells. Nat Commun 8 , 15287 (2017).
2. Takasugi, M. et al . Small extracellular vesicles secreted from senescent cells promote cancer cell proliferation through EphA2. Nat Commun 8 , 15729 (2017).