Figure 3.

Zeb2 5′-UTR contains an IRES element. (A) Diagram of the bicistronic plasmids, pRF and pRF-L (promoterless), used in these assays. (B) RWP-1 cells were transfected with pRF plasmid containing the indicated inserts. Results were normalized according to Renilla Luciferase and referred to the value obtained with empty pRF vector. The figure shows the average ± SD of four experiments performed in triplicate. (C) pRF and pRF +2359/+2998 plasmids were transfected to RWP-1 cells; RNA was collected 2 d after transfection and analyzed by RT–PCR with the oligonucleotides indicated in the diagram, corresponding to Renilla Luciferase (top), Renilla Luciferase and +2359/+2998 insert (middle), or Renilla Luciferase and Firefly Luciferase (bottom). (D) RWP1- cells were transfected with pRF-L and pRF-L +2359/+2998 plasmids and pRF containing just Renilla Luciferase. Expression of Renilla Luciferase was dependent on the expression of this plasmid; no activity was detected with pRF-L plasmid. Activity of Firefly Luciferase was normalized according to Renilla Luciferase and represented in respect to the value obtained with empty pRF-L plasmid. The figure shows the average ± SD of three experiments performed in triplicate.