FIG. 8.

Expression and functional activity of Rex-1 mutants. (A) Western blot of Rex-1 protein expressed from 293T cells transiently transfected with wt Rex-1 and deletion mutants, the P180Term L170Term mutant, and cDNA plasmids. Proteins were detected using rabbit Rex-1-specific antisera. wt p27 Rex is indicated, and the arrows identify the truncated Rex proteins. α, anti. (B) Functional activity of Rex-1 was determined by using the modified HIV p24 Gag reporter assay. 293T cells were transfected with 0.25 μg pcTat, 0.5 μg pcGagRxRE-I, 0.05 μg CMV-Luc, and 0.1 mg wt and mutant Rex-1 DNA. Forty-eight hours after transfection, cells were harvested and assayed for p24 Gag production. The values represent actual p24 Gag production from a representative experiment performed in triplicate. Error bars indicate standard deviations. (C) The half-lives of wt Rex-1 and the P180Term mutant were determined by pulse-chase experiments as described in Materials and Methods. Proteins were quantified at different time points using the Typhoon imaging system.