Figure 3.

Release of CD63 and CD9 in EVs requires VPS4 activity. (A) Top: Immunoblot of whole-cell lysate showing the GFP-VPS4BEQ expression induced by a 2 hr pulse of tetracycline. Middle and Bottom: Immunoblot of ESCRT-III protein CHMP4A in soluble and insoluble fractions separated by centrifugation at 17,000 × g. Pellets were resuspended in the same volume as supernatants, and equal volumes of each were loaded. (B) Cell lysate from 0.05 × 10⁶ cells and 100k EVs from 10 × 10⁶ cells as indicated were immunoblotted for CD63, CD9, and calnexin. (C) Quantitation of CD63 and CD9 on immunoblots from four independent experiments comparing 100k EVs from control uninduced or VPS4EQ-expressing HEK293 cells. The lines indicate mean. (D) CD63, CD9, and syntenin in EVs were collected by PEG/ConA precipitation. Each lane represents a technical replicate prepared from one-third of the total culture supernatant; for quantitation, these were averaged to represent a single experiment. (E) Quantitation of CD63, CD9, and syntenin on immunoblots from two (syntenin) or three (CD63 and CD9) independent experiments comparing PEG/ConA EVs from control uninduced or VPS4AEQ-expressing HEK293 cells. The lines indicate mean.