Figure 4

Mutational analysis of Rex-1 phospho-specific mutants. (A) The functional activity of either wtRex-1 or Rex-1 mutants, as indicated, was determined using the modified HIV p24 Gag reporter assay. The specific amino acid substitution for each Rex-1 mutant is shown. 293T cells were transfected with 0.25 μg pcTat, 0.5 μg pCgagRxRE-I, 0.05 μg CMV-luc, and 0.1 μg of wtRex-1 or Rex-1 mutant plasmids. At 24 h post-transfection, cells were harvested and assayed for p24 Gag. The values represent actual p24 Gag production from a representative experiment performed in triplicate. Error bars indicate standard deviations. T, threonine; S, serine; A, alanine; D, aspartic acid. (B) Western blot analysis of wild-type and Rex-1 mutants. Whole cell lysates normalized for transfection efficiency were subjected to Western blot using rabbit Rex-1-specific antisera. Rex-1 is indicated. (C) To determine the subcellular localization of the Rex-1 mutants, HeLa-Tat cells were transfected with 1 μg of a control plasmid, wtRex-1, or various Rex-1 mutants. At 24 hours post-transfection, cells were stained with rabbit Rex-1-specific antisera (Green). Nuclei were stained with DAPI (Blue).