FIG. 3.

Recombinant HIV-1 Nef protein-mediated apoptosis of human brain MVECs. Representative TUNEL staining of MVECs exposed to GST- and baculovirally expressed HIV-1 Nef protein is shown. MVECs were exposed to either GST protein or HIV-1 Nef protein expressed from Sf9 insect cells for 72 h. The concentrations of protein used were 10, 50, and 100 ng/ml. As a positive control, MVECs were exposed to DNase at a concentration of 10 μg/ml for 10 min at room temperature. TUNEL assays were performed with the in situ cell death detection kit, TMR Red (Roche Diagnostics). The cells were analyzed by fluorescence microscopy (Olympus System microscope, model BX60, with fluorescence attachment BX-FLA). These results are representative of those from experiments performed in triplicate and repeated at least twice.