Fig. 5. Ark1.PkC levels do not fluctuate as cells progress through the cell cycle. A cell-cycle synchronised population of strain IH2036 was generated by elutrient centrifugation at 25°C in rich YES medium. The small G2 cells were harvested at the beginning of the experiment and samples were taken every 20 minutes to score the septation index (A) and for western blot analysis of whole cell extracts (B). The blot was cut in two and the top portion was probed with antibodies to a tubulin (TAT1) to act as a loading control for the Ark1.PkC blot (below). The position of the peaks seen in the septation index are indicated by asterisks above the septation profile and the western blot. Quantitation of the ratio of the TAT1 to Ark1.PkC signals for two independent blots led to the conclusion that there is no great fluctuation in Ark1.PkC levels as cells progress through the cell cycle (data not shown). (C) Ark1 protein levels remain constant after arrest in the G1 phase of the cell cycle. Extracts were prepared from cells which were manipulated to be in distinct cell cycle stages. IH2117 was blocked at 37°C for 4 hours 15 minutes and released to 25°C for 45 minutes (mitotic sample). In this mitotic sample 60% of the cells had either condensed chromosomes or were binuclear without septa. A culture of IH2116 was split in two and one half was incubated at 37°C for 5 hours 30 minutes to arrest cell cycle progression at the cdc10 execution point (G1-phase sample). (D) Quantification of the bands shown in C. In each case the intensity of the Tat1 signal in the upper panel was divided by the intensity of the Ark1.PkC signal in the lower panel.