FIGURE 2.

Plk3 phosphorylates HIF-1α in vitro. A, recombinant His₆-Plk3 was expressed in Sf9 cells and purified using the Ni-NTA affinity approach as described under “Experimental Procedures.” Mock purification was carried out using Sf9 control cell lysates. Purified His₆-Plk3 was confirmed by Coomassie Blue staining. B, Sf9 cell lysates infected with or without His₆-Plk3 baculovirus, along with eluted proteins, were blotted with Plk3 antibody. C, different amounts of Plk3 were assayed for kinase activity toward casein in the kinase reaction supplemented with [γ-³²P]ATP. A representative autoradiogram is shown. D, in vitro kinase assays were carried out in the presence of [γ-³²P]ATP, as well as the various components, as indicated. Wort stands for wortmannin. A representative autoradiogram is shown.