p62 is secreted within the lumen of immunoisolated exosomes. (A) Domain architecture and schematic illustrating the membrane topology of HA-CD63-mEGFP^ECL1 when inserted into the limiting membrane of endosomes (left) and exosomes (right). mEGFP should be quenched within the acidic lumen of late endosomes. (B) Immunoblot analysis of MDA-MB-231 cells expressing HA-CD63-mEGFP^ECL1. (C) Fluorescence microscopy of MDA-MB-231 cells expressing HA-CD63-mEGFP^ECL1 upon treatment with either DMSO or BafA1 (100 nM) for 16 h. Green: HA-CD63-mEGFP^ECL1; magenta: LysoTracker. (D) Immunoblot analysis of a PNS input and late endosome IPs from lysed HA-CD63-mEGFP^ECL1 cells. (E) Schematic illustrating the procedure to immunoisolate exosomes from HA-CD63-mEGFP^ECL1 cells. (F) Immunoblot analysis of a HSP input and exosome IPs from the conditioned medium of HA-CD63-mEGFP^ECL1 cells. (G) Immunoblot analysis of an extracellular HSP input, flow-through, and anti-GFP IPs from the conditioned medium of HA-CD63-mEGFP^ECL1 cells. (H) Immunoblot analysis of proteinase K protection assays performed on immunoisolated exosomes to assess whether the indicated proteins are sequestered within the lumen of exosomes. TAMRA-labeled α-synuclein was added as an extravesicular spike to confirm complete proteolysis. (I) Quantification of the proteinase K protection experiments from Fig. 4 H (n = 3). Source data are available for this figure: SourceData F4.