Extended Data Fig. 2: Density-related expression of the EV markers CD63 and CD81 in capsid+/ stabilizer+ eraEVs.
(a) Western blot analysis illustrating CD63 and Arc expression across various EV subpopulations, differentiated by density through density gradient centrifugation. The 70% sucrose cushion layer—representing the highest density—displayed a marked increase in both CD63 and Arc. Equal amounts of total proteins were loaded across all lanes. Displayed is one representative trial out of three independent experiments. (b) Immunomagnetic positive selection was conducted to isolate CD81+ EVs from filtered supernatant cell culture media laden with both control and engineered EVs. Following isolation, the CD81+ EVs were lysed to facilitate protein quantification and subsequent western blot analysis. (c) Utilizing the Pierce 660 nm Protein Assay, total proteins harvested from CD81+ EVs were quantified. Notably, there was a 2.3-fold upsurge in the protein count extracted from CD81 + /Arc+ engineered EVs in contrast to the capsid‒ control. (d) Western blotting of the CD81 + EV lysates affirmed the presence of Arc proteins. The absence of reducing agents in the sample buffer was deliberate since detection antibodies for CD9/CD63/CD81 often identify the disulfide bond associated with antigen epitopes. Under non-reducing conditions, substantial Arc oligomers are evident. Notably, despite comparable levels of monomer Arc, the quantity of Arc oligomers was significantly amplified in stabilizer+ EVs, underscoring the stabilization effect imparted by the A5U motif. Owing to our exclusion of an Arc knockout cell line, endogenous Arc was discernible in the no-transfection control set. Abbreviations: NT - no transfection control; AA - Arc/A5U-GFP; AG - Arc/GFP. (e-f) Quantitative breakdown depicting the distribution of Arc protein monomers and oligomers in each sample set, as determined through Western blot analysis. The figure presents one representative trial from three independent repeats. For uncropped western gels, refer to SD_ED_FIG2.
