FIG. 7.

Phosphorylation of recombinant P by Akt1. Recombinant PIV5 P with a six-His tag at its N terminus was purified from bacteria as described in Materials and Methods and used in in vitro kinase assays with activated Akt1. (a) Phosphorylation of the recombinant P by Akt1 in vitro. P, Akt1, or P plus Akt1 was mixed with [γ-³²P]ATP, and the reaction mixtures were resolved by SDS-PAGE. Phosphorylated proteins were detected by phosphorimagery. Shown on the right are the relative mobilities of Akt1 and P. (b) Phosphorylation of recombinant P immunoprecipitated with P-specific antibody by Akt1. The recombinant P protein purified from bacteria was incubated with P-specific antibody P282, Pk, or control antibody mouse immunoglobulin G and washed with immunoprecipitation buffer as described in Materials and Methods. The precipitated products along with the beads were incubated with activated Akt1 and [γ-³²P]ATP. The products were resolved by SDS-PAGE and visualized as described above.