Fig. 5

CLN-5⁺ expression in endothelial-derived EVs. a, b Western blot analysis of CLN-5 in EVs from supernatants of TNF-α-treated BMEC cultures. Total EVs (a) and exosome- and microvesicle-size EVs separated by differential ultracentrifugation (b), showing a 23 kDa molecular weight band consistent with the molecular weight of CLN-5. c FACS analysis of total EVs isolated from blood plasma of Tie-2-eGFP-CLN-5 mice with EAE, showing eGFP-CLN-5⁺ EVs alone (left) or eGFP-CLN-5⁺ EVs co-stained with PKH-26 dye to label all (eGFP-CLN-5⁺ and eGFP-CLN-5⁻) EVs (right), in the double-positive event gate. Approx. 0.4% of all blood-derived EVs are shown as eGFP-CLN-5⁺ PKH-26⁺. Nano-fluorescent-size standard beads (not shown) were used for setting the gates