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Western blot analysis shows SAG21-YFP and APS1-YFP proteins were not detectable in the extracellular fractions, including apoplastic wash fluids (AWFs), the P100 EV fraction (EVs), or the supernatant of the P100 fraction (S). As a positive control, annexin1 (ANN1)-YFP-tagged protein was secreted into AWFs and present in EVs. The abundantly secreted pathogen-related protein1 (PR1), absent in EVs, was used as a secretion control. TET8 native protein was used as a marker for EV containing fractions. S, supernatant after 100,000 x g centrifugation.

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