Fig. 7. 100k protein binds the C-terminus of eIF4G and evicts Mnk1 protein from eIF4F complexes in vitro. (A) Soluble recombinant proteins were purified from bacteria corresponding to full-length GST–100k, GST–Mnk1 and fragments of eIF4G for the N-terminus (N4G), middle (M4G) and C-terminus (C4G). It was not possible to isolate sufficient amounts of full-length eIF4G due to degradation. GST was removed from eIF4G by thrombin digestion. Equal amounts of purified eIF4G proteins (2 µg) and GST–100k or GST–Mnk1 proteins (0.3 µg) were incubated in vitro, GST fusion proteins were recovered by glutathione–Sepharose chromatography and bound proteins identified by SDS–10%PAGE and immunoblot analysis with specific antibodies to the eIF4G fragments, or to GST (for Mnk1 or 100k). (B) 293 cells were transfected with plasmids expressing GST or GST–Mnk1, extracts were prepared and equal amounts of eIF4F isolated by immuno precipitation of eIF4G (eIF4G IP). Equal amounts (2 µg) of purified recombinant GST–100k or GST fusion proteins were incubated in vitro with eIF4F complexes, and associated proteins detected by immunoblot analysis as shown. Equal amounts of whole cell lysates (‘lysate’) were resolved in lanes 4 and 5 to establish protein levels.