FIG. 2.
Efficient expression and functional activity of Rex-2. (A) Illustration of the S-tagged Rex-2 (S-Rex-2) expression vector construct (not drawn to scale). (B) Western blot of S-Rex-2 protein expressed from 293T cells transiently transfected with Rex-2 cDNA plasmids. Proteins were detected using rabbit Rex-2-specific antisera. Wt p24Rex and p26Rex are indicated, and the arrowheads identify S-tagged Rex-2 proteins. (C) The functional activity of S-Rex-2 was determined using the modified HIV p24 Gag reporter assay. 293T cells were transfected with 0.25 μg pcTat, 0.5 μg pCgagRxRE-II, 0.05 μg CMV-luc, and 0.1 μg of wt Rex-2 or S-Rex-2 plasmids. Twenty-four hours posttransfection, cells were harvested and assayed for p24 Gag. The values represent actual p24 Gag production from a representative experiment performed in triplicate. The error bars indicate standard deviations. (D) Affinity purification of S-tagged Rex-2 from mammalian cells. 293T cells were transfected with S-Rex-2, purified with S protein-agarose beads, eluted and resolved by SDS-PAGE analysis, and detected by Coomassie blue staining.