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Fig. 8. Protein-kinase activity of Nak1/Orb3 in vitro. (A) Nak1/Orb3-dependent kinase activity throughout the cell cycle. Samples were taken from cultures synchronized by cdc25-22 block-and-release at the time points indicated and kinase assays were performed with casein as a substrate. Synchronous cdc25-22 cells expressing untagged Nak1/Orb3 were used as a control. Protein levels of precipitated Nak1/Orb3-Myc were checked by western blotting and immunodetection with anti-Myc antibodies (data not shown). (B) Kinase activity of Nak1/Orb3-Myc samples (closed circles) and of untagged control samples (open circles) was quantified using a phosphor imager. Septation index (closed triangles). (C) Temperature-sensitive kinase activity of Nak1/Orb3-167/Myc in vitro. Kinase assays were performed at 25°C and 37°C, with samples taken from asynchronous cultures of wild-type cells (WT) expressing untagged Nak1/Orb3, tagged wild-type Nak1/Orb3 or tagged mutant Nak1/Orb3-167. Protein levels of precipitated (prec.) Nak1/Orb3 and of total Nak1/Orb3 and ${alpha}$ -tubulin in the extracts were checked by western blotting and immunodetection.

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