FIG. 6.
Requirement of the C-terminal domain of HIV-1 IN for IN-Impα3 interaction. (A) Plasmids encoding either GFP, GFP-IN, the C-terminal deletion mutant GFP-IN1-212, or the N-terminal deletion mutant GFP-IN50-288 were each cotransfected with the T7-Impα3 expression plasmid into 293T cells, and the interaction of each form of IN with Impα3 was analyzed by anti-GFP immunoprecipitation followed by Western blotting (WB) with an anti-T7 antibody. (Top) Bound T7-Impα3 was detected by IP with an anti-GFP antibody followed by WB using an anti-T7 antibody. (Center) The immunoprecipitated GFP, wild-type GFP-IN, and GFP-IN deletion mutants were detected by WB with an anti-GFP antibody. (Bottom) T7-Impα3 expression was detected with an anti-T7 antibody. (B) Plasmids expressing wild-type GFP-IN or the different IN C-terminal deletion mutants, including GFP-IN1-212, GFP-IN1-250, and GFP-IN1-270, were each cotransfected with a T7-Impα3 expression plasmid into 293T cells, and the Impα3 binding of each IN mutant was analyzed by the co-IP assay as described in Materials and Methods. (Top) Bound T7-Impα3 was detected by IP with anti-GFP followed by WB using anti-T7 antibodies. (Center) The immunoprecipitated GFP, wild-type GFP-IN, and GFP-IN deletion mutants were detected by WB with an anti-GFP antibody. (Bottom) Total T7-Impα3 expression in the transfected cells was examined by Western blotting using an anti-T7 antibody. (C). Intracellular localizations of different GFP-IN mutants. HeLa cells were transfected with plasmids expressing GFP, GFP-IN, GFP-IN1-212, GFP-IN1-250, or GFP-IN1-270. 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).