Activity of HIV-1 Rev on the MMTV RmRE in human cells. A. Reporter activity of RevGFP on the RmRE in Jurkat T cells. Cells were electroporated with pHMRluc or pHMΔeLTRluc (1 μg) with either 20 μg of EGFP, RemGFP or RevGFP expression plasmids. Cytoplasmic extracts were prepared and analyzed for Renilla luciferase (Rluc) activity. Average luciferase values for each reporter plasmid in the absence of Rev or Rem have been assigned a value of 1, and the other samples are reported relative to this value after normalization for DNA uptake using a co-transfected pGL3 reporter plasmid expressing firefly luciferase. Standard deviations from the average of triplicate transfections are indicated. B. Reporter activity of RevGFP on the RmRE in 293T cells. Cells were transfected using calcium phosphate precipitation of DNA as described in the Methods section. Values are reported as described in panel A. C. Western blotting confirms similar expression of Rev and Rem. A Western blot of protein extracts from Jurkat cells is shown. The unfused GFP band is not visible in this portion of the gel. The upper panel shows reactivity with GFP-specific antibody; the lower panel shows equal loading of protein extracts using an actin-specific antibody. Size markers are given in kilodaltons.