Exosomes are small extracellular vesicles that are secreted from cells. To characterize exosome fraction marker proteins of the tetraspanin family in particular, CD9, CD63, and CD81 are routinely used. CD63 expression constructs were employed to investigate the influence of the large extracellular loop (LEL) of CD63 on sorting into exosomes. When the LEL of CD63 fused with mCherry was deleted, the protein was no longer found in the purified exosome fraction. This finding demonstrates the importance of the LEL sequence for the recruitment of CD63 into exosomes.
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