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. Author manuscript; available in PMC: 2010 Dec 8.
Published in final edited form as: Traffic. 2010 Jan;11(1):110–122. doi: 10.1111/j.1600-0854.2009.01006.x

Figure 2.

Figure 2

Nef chimeras in exosomes enter target cells. (A) Schematic overview of the exosome effector assay. Fusion proteins were expressed first in HeLa.CIITA cells. Two days later, exosomes were harvested as in Fig. 1 and then layered onto TZM-bl cells that harbor an integrated HIV-LTR-luciferase reporter gene. (B) Exosome effector assay. Effects of fusion proteins on the HIV LTR were determined first following their transient expression in TZM-bl cells: GFP (lane 1, white bar), Nef.GFP (lane 2, white bar with black stripes), Nef.Tat.GFP (lane 3, black bar) and Tat.GFP (lane 4, black bar with white stripes). Next, exosomes were harvested from transfected HeLa.CIITA cells and added to fresh TZM-bl cells for 48 hours: GFP (lane 5), Nef.GFP (lane 6), Nef.Tat.GFP (lane 7) and Tat.GFP (lane 8). Luciferase activity was measured at the end of the incubation. Presented are relative luciferase values to those with GFP or GFP exosome samples. Results are presented as the mean ±SD of three independent experiments. Expression of chimeras in HeLa.CIITA cells and their incorporation into exosomes were evaluated with anti-GFP antibodies by western blotting. Protein bands are labeled. Intracellular localization of the above-mentioned proteins was visualized by direct fluorescence in TZM-bl cells (green). White scale bar represents 20 μm.