fig4

Figure 4. Transient expression of Nef.GFP in h-microglia affects protein composition and buoyant density of extracellular vesicles. Total EVs were enriched by simple ultracentrifugation from media of 48 h old, transfected h-microglia cultures, either expressing Nef.GFP or not (control D2). Pelleted particles from both conditions were separated on a 20%-60% sucrose gradient, and precipitated fractions were analyzed by immunoblotting with antibodies directed against GFP, typical EV proteins (Hsc70, Flotillin, Tsg101, CD63, Annexin A2) and EV impurity marker Calnexin. CL: Cell lysate; EV: extracellular vesicles; Nef.GFP: Nef green fluorescent protein; h-microglia: human microglia; D2: day 2 post-transfection; Hsc70: heat shock cognate 70; Flotillin: flotillin protein; Tsg101: tumor susceptibility gene 101; CD63: cluster of differentiation 63; Annexin A2: annexin A2 protein; Calnexin: calnexin protein.