Activated AKT phosphorylates GATA-1S310. Purified recombinant constitutively active AKT (act), kinase inactive AKT (inact), or constitutively active SGK protein (act) (A) or immunocomplexes of Flag-tagged constitutively active AKT (AKT^*) or HA-tagged kinase-inactive mutant of AKT (AKT K-D) (B) were used in an in vitro kinase assay and incubated in the presence of [γ-³²P]ATP with GST-GATA-1 WT or mutants as substrates. Samples were resolved on SDS-PAGE, and ³²P-labeled proteins were detected by autoradiography (A-B). Half of the reaction was subjected to Western blot analysis using the indicated antibodies (B). (C) 293T cells were cotransfected with GST alone or GST-GATA-1 wild type or mutants and a Flag-tagged constitutively active AKT (AKT^*) or a dominant-negative AKT (HA-AKT DN), lysates were prepared 48 hours later and subjected to Western blot analysis by using the indicated antibodies.