Fig. 6. Ofs associates with an mRNA cap analog. (A,B) Western blots of S2R+ cells transfected with Flag-Ofs or Flag-eIF4G (A) or with GFP-Ofs (B) and incubated with either 7-m-GTP-Sepharose (7-methyl-GTP Sepharose) beads or glutathione-Sepharose beads (control). Relevant bands are indicated with an arrow (Ofs constructs) or arrowhead (Flag-eIF4G). Asterisks indicate cross-reacting bands. Input lanes represent 2.5% of total lysate; pulldown lanes represent 100% of protein recovered from the beads. (A) Both Flag-Ofs and Flag-eIF4G are pulled down along with eIF4E by 7-m-GTP-Sepharose beads but not by control beads. (B) Binding is not due to the Flag epitope, because GFP-tagged Ofs also associated with 7-m-GTP-Sepharose beads. All conditions were repeated at least three times with comparable results.