FIG. 3.

Specificity of A3H incorporation and subcellular localization. (A) HIV-1 virions (500 ng) were produced by transfection in the presence of A3H hapI-GKE, A3H hapII-RDD, A3G, or an empty pTR600 plasmid (100 ng). Virions were concentrated through a 20% sucrose cushion and dissolved in PBS alone, PBS with proteinase K, PBS with Triton X, and PBS with both proteinase K and Triton X. Virions were incubated at 37°C for 30 min. A3H and A3G expression levels (anti-FLAG) were assessed by Western blotting. HIV-1 p24 and gp120 were probed by using anti-HIV serum. A band corresponding to proteinase K is indicated by an asterisk. (B) Cellular A3H and A3G expression levels (anti-FLAG) were assessed by Western blotting. Detection of GAPDH served as a protein loading control. (C) 293T cells were transfected with N-terminally FLAG-tagged A3H hapI-GKE and A3H hapII-GKE, and the subcellular localization was assessed by confocal microscopy. Two different imaging settings were used since a higher offset was needed to capture the poorly expressed A3H hapI-GKE. Representative images are shown at a ×100 magnification. DNA was stained with Hoechst dye to visualize the nuclei.