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. 2010 Jun 16;84(17):8650–8663. doi: 10.1128/JVI.00508-10

FIG. 5.

FIG. 5.

Impα3 interacts with HIV-1 integrase (IN) in vitro and in cotransfected 293T cells and HIV-infected C8166 T cells. (A) (Left) Cell lysates from 293T cells expressing GFP or GFP-IN were incubated with either GST alone or a recombinant GST-Impα3 fusion protein. After GST pulldown, the bound proteins were analyzed by Western blotting (WB) with an anti-GFP antibody. (Right) Equal amounts of GST and GST-Impα3 were incubated with purified recombinant HIV-1 IN followed by GST pulldown and were analyzed on an SDS-PAGE gel by Western blotting with rabbit anti-IN antibodies. (B) Impα3 interacts with HIV-1 IN and Vpr in 293T cells. A full-length human T7-Impα3 expression plasmid was cotransfected with a plasmid expressing GFP, GFP-IN, MA-YFP, or Vpr-YFP into 293T cells. (Top) After 48 h of transfection, 90% of the transfected cells were lysed in 0.25% NP-40 buffer and were immunoprecipitated with a rabbit anti-GFP antibody. The immunoprecipitates were resolved using 10% SDS-PAGE, and the bound T7-Impα3 was detected by WB with an anti-T7 antibody. (Center) The presence of GFP-INwt/mut in immunoprecipitates was detected by an anti-GFP antibody. (Bottom) To check protein expression, the remaining 10% of the transfected cells were lysed with 0.5% NP-40, run directly on a 10% SDS-PAGE gel, and probed with anti-T7 antibodies. (C) HeLa cells were transfected with a GFP, GFP-IN, MA-YFP, or Vpr-YFP plasmid. After 48 h, cells were fixed and labeled with an anti-GFP antibody, and the nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Cells were then analyzed by fluorescence microscopy (with a 40× objective lens). (D) Impα3 interacts with HIV-1 IN in virus-infected C8166 T cells. Approximately 10 × 106 C8166 T cells were infected with an HxBru or HxBru-IN-HA virus. (Top) Seventy-two hours after infection, cells were lysed and immunoprecipitated with a rabbit anti-HA antibody, and the bound endogenous Impα3 was detected by Western blotting using rabbit anti-Impα3. The normal C8166 cell lysate was loaded as a positive control (PC). (Center) The immunoprecipitated HA-IN was also detected by WB using a mouse anti-HA antibody. (Bottom) Lysates from the infected C8166 cells were directly loaded onto an SDS-PAGE gel to detect HIV p24gag protein by WB using an anti-p24 antibody.