Fig. 3. C-Nap1 is phosphorylated during mitosis. (A) Alkaline phosphatase treatment enhances the western blot signal of mitotic C-Nap1. C-Nap1 was immunoprecipitated from asynchronously growing (asy) or nocodazole-arrested (noc) HeLa cells and treated with either active (+) or heat-inactivated (-) calf intestinal phosphatase (CIP). Samples were separated by 7.5% SDS-PAGE and probed by immunoblotting as indicated (IgG are shown to demonstrate equal loading). (B) In vivo labeling of C-Nap1 with [³²P]orthophosphate. C-Nap1 was immunoprecipitated from the indicated cell cycle stages, using C-Ab. Samples were separated by 7.5% SDS-PAGE, and gels exposed for autoradiography (top panel). In parallel, recovery of C-Nap1 was controlled by western blotting with C-Ab (bottom panel).