FIG 4.

S6K interacts with and phosphorylates eIF3 in vitro. (A) HEK-293T cells were transfected with constructs expressing HA-tagged eIF3f (3f), eIF3g (3g), GFP, or an empty vector. Whole-cell lysates (WCL) were subjected to immunoprecipitation with an anti-HA antibody. WCL and HA immunoprecipitates were processed for Western blotting with the indicated antibodies. IgB, light-chain immunoglobulin. (B) Kinase assays were performed with recombinant His-tagged S6K1 (HIS-S6K) and RRL-purified eIF3 (eIF3). Proteins were incubated for 20 min with [γ-³²P]ATP in the presence of 100 nM DG2. Samples were subjected to SDS-PAGE and transferred onto nitrocellulose membrane, followed by Western blotting and autoradiography.