Figure 5.

N interacts with the pre-initiation complex. (A) N was incubated with rabbit reticulocyte lysates and recovered with Ni-NTA beads. The bound material was eluted from the Ni-NTA, RNA was purified, and 18S rRNA was quantified using real-time RT–PCR. The leftward graph depicts the relative amount of 18S rRNA associated with Ni-NTA beads in the absence and presence of N. The rightward graph depicts an analogous experiment carried out with 293 cells that were transfected with either an N-expressing plasmid or its parental vector, as a negative control. N was recovered from the lysates of transfected cells using Ni-NTA and 18S rRNA that co-purified with N quantified by real-time RT–PCR. (B) A set of western blots to examine the association of peptide constituents of the 43S pre-initiation complex, and the eIF4F cap-binding complex, that co-purify with N. N was expressed by transfection, isolated from the lysates of these cells using Ni-NTA columns, bound material was recovered and subjected to western blot analysis with primary antibodies as indicated. Peptides that co-purify with N (bound), or that flow through the column are indicated.