Figure 1. Annexin-containing EVs are... | Rockefeller University Press

Figure 1.

$Annexin-containing EVs are distinct from exosomes. (A) Immunoblots show the distribution of EV markers across an iodixanol gradient of the conditioned medium 100k × g pellet fraction. Samples were taken from low (F2—Fraction #2) to high density (F14—Fraction #14). (B) Immunoblots show depletion of annexins from exosomes after immunoprecipitation with anti-CD63 beads from the conditioned medium 100k × g pellet fraction (W1—wash #1; W2—wash #2). (C) Immunoblots show degradation of EV markers in the conditioned medium 100k × g pellet fraction after treatment with indicated combinations of proteinase K (ProK) and 0.1% TX-100. For EGTA treatments, 5 mM EGTA was introduced into the medium prior to centrifuging the conditioned medium at 100k × g. (D) Schematic illustrating the separation of EVs for quantitative proteomics using sucrose step gradients is shown. (E) Volcano plot shows enriched proteins in the high buoyant density fractions (red) relative to the low buoyant density fractions (green). P values were calculated using a t test. Source data are available for this figure: SourceData F1.$

Annexin-containing EVs are distinct from exosomes. (A) Immunoblots show the distribution of EV markers across an iodixanol gradient of the conditioned medium 100k × g pellet fraction. Samples were taken from low (F2—Fraction #2) to high density (F14—Fraction #14). (B) Immunoblots show depletion of annexins from exosomes after immunoprecipitation with anti-CD63 beads from the conditioned medium 100k × g pellet fraction (W1—wash #1; W2—wash #2). (C) Immunoblots show degradation of EV markers in the conditioned medium 100k × g pellet fraction after treatment with indicated combinations of proteinase K (ProK) and 0.1% TX-100. For EGTA treatments, 5 mM EGTA was introduced into the medium prior to centrifuging the conditioned medium at 100k × g. (D) Schematic illustrating the separation of EVs for quantitative proteomics using sucrose step gradients is shown. (E) Volcano plot shows enriched proteins in the high buoyant density fractions (red) relative to the low buoyant density fractions (green). P values were calculated using a t test. Source data are available for this figure: SourceData F1.

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