Figure 6.

Site B is a bona fide stability element. (a) Jurkat/D1.1 cells were transiently transfected with either pRLABC (top panel) or pRL-ΔB (lower panel) and treated with 5,6-dichlorobenzimidizole 1-m-d-ribofuranoside (DRB) over a 4-hr time–course. Total RNA was isolated from cells treated with DRB for 8 hr and Renilla luciferase (lanes 1–5) or control β-actin (lanes 6–10) expression was analysed by semi-quantitative reverse transcription–polymerase chain reaction (RT-PCR). The RT-PCR was conducted with a concentration of complementary DNA that was previously determined to be in the linear range of amplification. (b) Time-points from three independent transfections were averaged and the SEM was calculated. Curve-fitting was performed by non-linear regression. (c) Schematic representation of the pRL-D construct and a graph of the fold induction of Renilla luciferase activity for each construct over the control pRLSV40 plasmid. Levels of activity for each construct were normalized to the firefly luciferase activity. Results represent the average of at least three independent experiments ± SEM. (d) Schematic representation of the pRL-A5′ construct and DRB assays using RNA from transiently transfected cells expressing pRL-1348-E1, pRLSV40 and pRL-A5′. RNA was isolated at four different time-points. The results represent the average ± SEM of two independent transfections.