Figure 4

The insulin-stimulated, rapamycin-sensitive, association of eIF4G with eIF3 does not require eIF4E binding to eIF4G. (A) FLAG-eIF4E was coexpressed in 293T cells with HA-tagged forms of either wild-type (WT) eIF4G or a mutant eIF4G (AA) rendered deficient in eIF4E binding. eIF4E was partially purified from extract samples (750 l) by using m⁷GTP agarose. Proteins were eluted from the beads and subjected to SDS–PAGE along with samples (15 l) of extract before immunoblots were prepared with antibodies to either the HA or FLAG epitopes. (B) Proteins were overexpressed as described above before immunoprecipitations were conducted with FLAG antibodies. Immunoblots were prepared with antibodies to either the HA or FLAG epitopes. (C) HA-tagged eIF4G (WT) and eIF4G (AA) were overexpressed in 3T3-L1 adipocytes by using adenoviral vectors. After 24 h, the cells were incubated without and with rapamycin and/or insulin as described above. Immunoprecipitations were conducted with nonimmune IgG or eIF3b antibodies. Immunoblots were prepared with eIF3b antibodies and antibodies to the HA epitope. (D) The relative amounts of HA-tagged forms of eIF4G that coimmunoprecipitated with eIF3 were expressed as percentages of the maximum HA signal recovered in each experiment. Mean valuess.e. from five experiments are presented.