Akt1 phosphorylates human and mouse p27. A, Purification of His-tagged p27 from E. coli. Upper panel: Aliquots from indicated steps of the purification protocol (see Methods) were separated by SDS-PAGE and visualized by Coomassie blue staining. Bottom panel: Indicated forms of His-tagged p27 were purified as described in Methods and their concentration compared by SDS-PAGE followed by Coomassie staining. B, Constitutively active Akt1 phosphorylates p27 lacking T157. An in vitro kinase assay was performed as described in the Methods by incubating commercial recombinant Akt1(prep1) (see Methods) and [32P]ATP with the indicated substrates. Samples were then separated by SDS-PAGE and visualized by autoradiography. Lane 1 is a negative control lacking Akt1 substrate, while lane 2 shows Akt1 phosphorylates its well-known substrate GSK. Lane 3–5 show Akt1 phosphorylates human wild type p27, mouse p27, and hp27T157A equally well.