FIG. 3.

Reductions in the expression of p54nrb, PSF, YB-1, and GRSF-1 correlate with decreases in IRES activity in cultured cells. Cells were transfected with plasmids that reduce the expression of p54nrb, PSF, GRSF-1, or YB-1 and then transfected with the plasmids harboring the c-, L-, or N-myc (pRMF, pRLsF, pRNF, respectively); Apaf-1 (pRAF); or BAG-1 (pRBF) IRES. Cells were harvested and lysed, and a proportion of the resulting material was immunoblotted using antibodies against small interfering YB-1 (si-YB1) (A), p54nrb (B), PSF (C), or GRSF-1 (D); actin was used as a loading control (Con). In each case, the level of the protein was reduced to less than 20% of the value in the control cells. The samples were then assayed for firefly and Renilla luciferase activities, and the relative IRES activity was calculated as firefly activity relative to the transfection control β-galactosidase. All experiments were carried out in triplicate and performed on at least three independent occasions. All three myc IRESs were affected by a reduction in the levels of these four proteins, although the greatest decrease was observed when the level of YB-1 was reduced, causing the activity of all three IRESs to be reduced by 95%. In contrast, only a small reduction in Apaf-1 IRES activity was observed in cells with a decreased expression of p54nrb, and no effect was observed in BAG-1 IRES activity.