Objective: The authors may include “packaging and localization of Annexin A2” instead of just “packagin of Annexin A2” as the article deals with the localization on the surface of EVs. Introduction contains only one sentence about Annexin A2. It is an important protein with lots of literature available on it. I would ask if a bit more detail about Annexin A2 function and relevance could be provided for example its involvement in endocytosis, ion channel formation etc. Fig 1C: In this figure authors just show the blot for CD63 a well-known exosomal marker. I would ask if the authors could also show the blot for cell lysate containing CD63 and the expected enrichment of CD63 in exosomal fraction. In the same figure I would ask to include negative controls like calnexin to conclusively show the purity of exosomal preparation.
Fig 1E: This figure is one of the main figures of the article. The authors have used EDTA as a calcium chelator. I would suggest the use of other chelators like BAPTA-AM, EDS to conclusively show the shedding of Annexin A2 from the surface is just not an EDTA specific effect.
It is known that Annexin A2 is presence on the cell surface. So its presence on the surface of EVs could originate from microvesicles and not from exosomes. I would request the authors to discuss this possibility in a bit more detail.
Is this phenomenon just specific to Annexin A2? If the authors could discuss the localization (surface or cytosolic) of other proteins that are lacking signal sequence but are enriched in exosomes, it would enhance the quality of discussion. For this part no experimentation is required, just discussion to enhance the relevance of this interesting finding with respect to other proteins.