Figure 5.

GSK-3 directly phosphorylates NPM1. A, multiple phosphorylated forms of NPM1 were detected by Western blotting of wild-type and Gsk3 DKO cell lysates resolved by Phos-tag gel (top). NPM1 migrates as a single species in standard electrophoresis (middle). Bottom, β-tubulin loading control. B, Gsk3 DKO cell lysates were added to an in vitro protein kinase reaction with recombinant GSK-3 with or without ATP for 1 h. The reaction was then resolved by Phos-tag gel, and NPM1 was visualized by Western blotting. A slower-migrating species detected only in the presence of ATP, representing a GSK-3–phosphorylated form of NPM1, is indicated by the red arrow. C, GSK-3 phosphorylation of recombinant NPM1 in vitro resolved by Phos-tag (top) and standard electrophoresis (bottom).