Figure 4.

Zeb2 5′-UTR contains an IRES element. (A) A bicistronic RNA containing +2359/+2998 insert placed between Renilla and Firefly Luciferase was synthesized in vitro and capped as indicated in the Materials and Methods. This bicistronic RNA and a control without insert were transfected to RWP-1 cells ectopically expressing Snail1 (B), or to control RWP-1 cells (C). Renilla and Luciferase activities were determined after 12 h and referred to the value obtained for Renilla Luciferase in the transfection of capped RNA. The figure shows the average ± SD of three experiments performed in triplicate. (D) RNA was isolated from RWP-1 Snail1 cells transfected with the uncapped bicistronic transcripts 6 h after transfection, or from cells transfected with pRF-+2359/+2998 plasmid. Five micrograms of RNA were analyzed by Northern blot using a random prime [³²P]-labeled probe corresponding to Firefly Luciferase coding region. Migration of the 28S and 18S ribosomal RNAs is indicated. The arrows indicate the migration of the transcripts corresponding to RLuc-+2359/+2998-FLuc and FLuc.