Figure 5.

Overexpression of p100 or SN-like domains increases the activity of luciferase construct containing AT1R 3′-UTR. A schematic representation of luciferase constructs used is shown. (A) Overexpression of p100 increases the expression of luciferase construct containing 3′-UTR. COS-1 cells were transfected either with a luciferase coding region (Luc) or with Luc(3′-UTR) construct where luciferase gene was fused with full-length AT1R 3′-UTR, and co-transfected with empty pSG5 vector, or pSG5 plasmid expressing p100-Flag, together with renilla luciferase. Cells were either lysed and assayed for firefly and renilla luciferase activities (upper column) or subjected to RNA extraction, followed by RT–PCR and quantitative luciferase-PCR (lower column). Results are shown as a mean of three independent experiments and SD. The values corresponding to transfections with Luc construct together with empty pSG5 vector was set to one. A representative anti-Flag western blot of p100-Flag expression is shown (bottom panel). (B) SN-like domains increase the activity of Luc (1–300) construct. COS-1 cells were transfected with Luc(1–300) construct together with empty pSG5 vector, or expression constructs for p100-Flag, SN-Flag or TD-Flag together with renilla luciferase. Cells were lysed and assayed for firefly and renilla luciferase activities. Results are shown as a mean of three independent experiments and SD. Value corresponding to transfection with Luc(1–300) construct together with empty pSG5 vector was set to one. An anti-Flag western blot shows the relative expression levels of p100, SN-like domains and TD.