NefG3C-GFP incorporates at high levels in HIV-1 based VLPs. Western blot analysis for the virion incorporation of NefG3C-GFP as compared with wt Nef and wt Nef-GFP (A) or with GFP-Vpr (B). 293T cells were transfected with the pCMVΔR8.74 HIV-1 packaging vector together with vectors expressing either wt Nef, wt Nef-GFP, or GFP-Vpr in 1:1 molar ratio. Forty-eight hours later, supernatants were harvested, and the VLPs purified on a 20% sucrose cushion. Then, the VLP concentrations were measured by a quantitative anti-HIV-1 CAp24 ELISA, and 100 ng of VLPs were analyzed by Western blot using a 1:1000 dilution of the ARP444 anti-Nef polyclonal Abs, a 1:200 dilution of the 6.2 anti-Nef mAb (A), or a 1:500 dilution of an anti-GFP mAb (B). As negative control (Ctrl-), 100 ng of empty VLPs were used in both cases. In addition, 50 μg of the lysates from the producer cells were probed for the expression of HIV-1 CAp24, Nef-, and GFP-related products. (C) Anti-Nef Western blot analysis of the indicated volumes of 100-fold concentrated and purified supernatants from 5 × 106 293T cells transfected with the NefG3C-GFP expression vector alone or together with the pCMVΔR8.74 HIV-1 packaging vector in a 1:1 molar ratio. In the here presented experiment, the virion concentration in the preparation from the cells transfected with the HIV-1 packaging vector was 3 ng/μl of CAp24. Also, 50 μg of the lysates from transfected cells were analyzed for the NefG3C-GFP expression. Data are representative of 9 (A), 3 (B), and 2 (C) independent experiments. The migration of major viral products are indicated on the left side, whereas the molecular marker sizes are reported on the right.