Fig. 4. Hsps are located within the exosome lumen. (a) Purified stock exosomes were analysed by western blot for induced hsp expression. Aliquots of these exosomes were subsequently immobilised onto latex beads and surface-hsp expression was analysed by (b) flow-cytometry; filled histograms represent exosome-bead complexes stained with irrelevant-isotype control antibody, unfilled histogram represent antibody staining as indicated. (c) Remaining exosome-bead complexes, not stained with antibodies, were boiled in SDS-sample buffer and analysed by western blot. (d) The remainder of the initial exosome stock (intact exosomes, not immobilized onto latex beads) was immunoprecipitated with antibodies as indicated and the relative quantity of precipitated exosomes determined by western blot, stained for MHC class I. Although exosomes were effectively precipitated with MHC class II antibodies, no bands were seen with hsp-specific antibodies. These data indicate that hsps are detectable following exosome disruption, but are not present on the exosome surface.