Fig. 1. Cdc8 protein levels do not fluctuate during the cell division cycle. (A) A novel anti-Cdc8 antibody was characterised by western blot of protein extracts from wild-type strains containing either the multicopy plasmid pREP41 (empty vector) or pREP41cdc8, or from a wild-type strain in which the plasmid pINT41gfp-cdc8 had been integrated at the leu1⁺ locus. All cells were grown in de-repressed conditions (– thiamine). An extract of wild-type cells containing the empty vector, pREP41, shows a Cdc8 band that migrates at 27 kDa. The intensity of the band increased in wild-type cells in which cdc8⁺ was additionally expressed from a plasmid under the control of the nmt41 promoter (pREP41cdc8⁺). Extracts from cells expressing chromosome integrated gfp-cdc8 showed an additional GFP-tagged Cdc8 at 54kDa, the intensity of which was comparable to the endogenous Cdc8 band. The same GFP-Cdc8 band was seen when the membrane was probed with anti-GFP antibody (B). (C) The same membrane probed with TAT1 to demonstrate equal loading of protein. A population of DPM126 cells were synchronised using transient block and release of the cdc25-22 allele. Two cell cycles were followed and samples were taken every 20 minutes for western blot analysis and for scoring the appearance of actin rings and septation index at each time point. Positions of molecular mass markers (in kDa) are indicated. (D) The proportion of cells possessing cytokinetic actomyosin rings (O) or a septum (+) at each time point over the two cell cycles. A western blot of whole cell extracts from each time point was probed with both Cdc8 anti-sera (E) and TAT1 antibody (F). Cdc8 migrates as a constant doublet throughout the cell cycle. Quantification of the ratio of TAT1 to Cdc8 doublet signals were analysed from three independent experiments and revealed no fluctuation in Cdc8 levels as cells progress through the cell cycle.