fig8

Figure 8. Kinetics of extracellular release of Nef from h-microglia infected with VSV-G pseudotyped HIV-1. Immunoblot analysis of cell lysates and extracellular particles (EVs + VIR) from h-microglia cultures infected with VSV-G pseudotyped NL4-3 and collected at the indicated time points. Extracellular particles were enriched from culture media by ultracentrifugation through a sucrose cushion. Antibodies directed against viral proteins (gp120, Nef, p24), typical EV proteins (Alix, Flotillin), marker of efficient gradient separation (AChE) and impurity markers (Calnexin, Albumin and Cytochrome c) were used. CL: Cell lysate; EV: extracellular vesicles; VIR: virus; AChE: acetylcholinesterase; Nef: negative factor; h-microglia: human microglia; VSV-G: vesicular stomatitis virus glycoprotein; HIV-1: human immunodeficiency virus type 1; NL4-3: HIV-1 strain NL4-3; gp120: glycoprotein 120; p24: HIV-1 core protein p24; Alix: ALG-2-interacting protein X; Flotillin: flotillin protein; Calnexin: calnexin protein; Albumin: serum albumin; Cytochrome c: cytochrome c protein.