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Figure 8. p17-peptide incorporation in MSC-EVs. (A) Representative dot plots from flow cytometry analysis of EVs; samples were analyzed in the FITC channel to detect CD63-p17-peptide-GFP-positive EVs; (B) The bar graph summarizes the relative abundance of GFP-EVs across the different CD63-based p17-derived peptide constructs, indicating construct-dependent variability in GFP signal while confirming the presence of GFP-positive EV subpopulations; (C) Western blotting of MSC-derived EVs from UT cells and cells expressing CD63-GFP constructs. GFP blotting reveals the peptide-tagged CD63 constructs, migrating as a single ~75 kDa band for CD63-mut and as an additional ~40-45 kDa band for peptide-tagged variants. Alix and SDCBP blotting, together with CD81 dot blot, confirm the EV identity of the samples; GFP dot blot confirms the presence of engineered EVs and quantifies the GFP signal used in (D). Molecular weight markers are indicated on the left and expressed in kDa. Raw, uncropped blots corresponding to (C) are shown in Supplementary Figure 2F-K; (D) Bar histogram of the relative GFP abundance across constructs, expressed as the GFP/Alix ratio (mean ± SD). CD63: Cluster of differentiation 63; DB: dot blot; EV: extracellular vesicle; FITC: fluorescein isothiocyanate; GFP: green fluorescent protein; kDa: kilodalton; MSC: mesenchymal stromal cell; MW: molecular weight; SDCBP: syndecan-binding protein; SD: standard deviation; SSC: side scatter; UT: untransfected.