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

 

User Report

Akiko Takahashi

Foundation for Research on Cancer

Products

• ●Anti CD81 for Exosome Isolation, Human (Mouse) Unlabeled, 12C4（Cat.No. SHI-EXO-M03）

Experimental findings

Exosomes arise by the action of the ESCRT complex from vesicles contained in multivesicular endosomes. These intraluminal vesicles are secreted as extracellular vesicles with diameter of 50-150 nm. Recently, with the discovery that exosomes contain proteins, lipids and nucleic acids, much attention has focused on their function as intercellular communication vehicles.

We are in the process of analyzing the senescence-associated secretory phenotype, characterized in part by elevated secretion of inflammatory cytokines and chemokines. Senescent cells also elevate their secretion of exosomes (Reference 1). We induced cellular senescence by serial passage of normal human fibroblasts (replicative senescence) and by introduction of the activated oncogene RasV12 (oncogene-induced senescence). Following 48 hours of incubation, exosomes in the conditioned medium were recovered by ultracentrifugation. The size distribution of the recovered extracellular material was characterized by Nanoparticle Tracking Analysis. The number of secreted exosomes was 30 to 50 times higher in senescent cells than young cells (Figure 1, top). To further compare senescent versus young exosomes we performed western blotting experiments using an anti-human CD81 monoclonal antibody (clone 12C4, used at 1:1000 dilution; from Cosmo Bio USA). The results showed an increase in the amount of CD81 protein recovered from senescent versus young exosomes (Figure 1, bottom). Exosomes recovered from senescent cells have also been observed to exhibit a growth-promoting effect on MCF-7 breast cancer cells (Reference 2).

The physiological role and diagnostic/therapeutic potential of exosomes are still incompletely understood. We are analyzing the detailed molecular mechanism by which normal and aged cells secrete exosomes, with the hope of clarifying the biological significance of exosomes. Furthermore, by studying the senescence-associated secretory phenotype, I hope to elucidate the roles that cancer exosomes play in age-related diseases.

Figures

Analysis of exosomes from young and senescent versions of human fibroblast cell line TIG-3 (from Reference 1).

Top: Relative number of exosomes recovered as determined by NTA (Nanoparticle Tracking Analysis). Bottom: Western blot detection of exosomal marker proteins.

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)