Figure 7.

The effect of a dominant negative mutant of eIF4A on translation. (A) schematic representation of the GFP-reporter genes is shown in the upper-left panel. The first has a long unstructured 5′ UTR, the second has a hair-pin structure within the 5′ UTR and the third has TISU in a context of a short 5′ UTR. HEK293T cells were transfected with these GFP reporters together with increasing amounts of eIF4A-dominant negative mutant (eIF4A-DN) or wild-type eIF4A-expression plasmids as indicated. The amount of expression plasmid was kept constant with the empty expression vector. RL under the CMV promoter was found to be refractory to eIF4A-DN (see Supplementary Figure S6) was used to normalize for transfection efficiency. GFP, eIF4A-DN and eIF4A expression were analyzed by immunoblot in which representative are shown on the upper right section. The asterisk in the eIF4A blot denotes a non-specific band. A graph representing the average of densitometric measurements of three independent transfection experiments is shown in the lower panel.