Figure 5

HIV-1 antisense gene proteins (HAPs) are translated within human cells. (A) Schematic diagram of recombinant vector containing the HIV-1 antisense gene sequences linked to carboxyterminal sequences encoding a FLAG epitope (HIV-AS-FLAG), or the same HIV-1 sequences in the opposite orientation linked to the same vector (REV). Landmarks in the HIV-1 LTR U3 region are not to scale. (B) HL2/3 cells were transfected with HIV-AS-FLAG, mock transfected (-), or transfected with the identical vector containing a luciferase gene linked to FLAG (+), and the cell lysates incubated on anti-FLAG immunoaffinity columns. Following incubation of the three lysates, each column was washed, and then "batch eluted". The eluates were then PEG concentrated and analyzed using Tris-tricine-urea SDS-PAGE with a mini-gel apparatus, followed by silver staining. MM is Multi-Mark multi-colored standard (Invitrogen). (C) HIV-AS-FLAG or REV (-) control transfected-cell lysates were incubated on anti-FLAG immunoaffinity columns, and the concentrated, pooled eluate fractions analyzed by Tris-tricine-urea SDS-PAGE (large gel apparatus) and silver staining. The HIV-AS-FLAG eluates were treated with DTT, with DTT and iodoacetamide (D/I), or received no treatment (NT), as indicated. E, empty; M, Mark 12 protein standard (Invitrogen). (D) Immunoblot analysis of HIV-AS-FLAG transfected cells. Cell lysates from HL2/3 cells transfected with HIV-AS-FLAG were affinity-purified over anti-FLAG M2 antibody resins, and the eluates were then concentrated (AS-FLAG). The eluates were split and subjected to either no treatment (NT) or treatment with DTT and Iodacetamide (D/I), and then analyzed using Tris-tricine-urea SDS-PAGE with a mini-gel apparatus, blotted to nitrocellulose membranes and probed with either AIDS sera, normal serum, or biotin-labeled mouse monoclonal anti-FLAG M2 antibody, as indicated. M, MultiMark multi-colored standard (Invitrogen). Molecular weights are in kilodaltons.