Density gradient analysis of wild-type, CNP-null, and PLP-null sEVs.

sEVs isolated from culture supernatants derived from equal numbers of wild-type, CNP-null, and PLP-null oligodendrocytes by differential ultracentrifugation followed by density gradient centrifugation (0.3–1.8 M sucrose gradient). Individual fractions were analyzed by Western blotting using established oligodendroglial sEV markers, PLP/DM20 (DM20 is a smaller isoform of PLP), Sirtuin-2, and Flotillin-1. Slight shifts in fractions between the markers indicate heterogeneity in sEV/exosome populations, which has been recognized previously [8]. Wild-type and mutant sEVs appear of similar density. Note that marker intensity appears weaker in mutant sEVs, reflecting a lower sEV yield from the same volume of starting material. Underlying images of blots can be found in S1 Images.

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