Akt1 phosphorylates p27S10. A, Determination of S10 phosphorylation by specific antibody. Recombinant Akt1 was incubated with the indicated forms of His-tagged p27 in the presence of non-labeled ATP. Samples were separated by SDS/PAGE and analyzed by western blot with the indicated antibodies. B, Alkaline phosphatase abolishes recognition of phosphorylated p27. hwtp27 was phosphorylated as in A and treated with or without alkaline phosphatase. Samples were then analyzed by western blot with the indicated antibodies. C, Akt1 phosphorylates serine 10 in mouse p27. Akt1 was incubated alone or with the indicated forms of p27 in the presence of non-labeled ATP. Total and phospho-S10 levels were analyzed by western blot as indicated. D, Phospho-peptide maps of human and mouse p27. Radiolabeled samples were analyzed as described in Figure 3A.