Fig. 7. Nak1/Orb3 protein levels in synchronized Nak1/Orb3-Myc cdc25-22 cells. The cells were blocked in G2 phase at 36.5°C for 3.5 hours and synchronously released into mitosis by cooling the culture to 25°C. Samples were taken every 20 minutes. (A) Septation index. The proportion of cells displaying a septum was determined for each time point. (B,C) Nak1/Orb3-Myc protein levels. Denatured protein extracts were prepared and equal amounts for each time point were separated by SDS-PAGE. The proteins were detected with monoclonal antibody 9E10 against the Myc tag. (B) Separation on a 4-12% acrylamide gradient gel. (C) Separation on a 8% acrylamide/0.1% bisacrylamide gel (left). Phosphorylated (Nak1/Orb3-P) and unphosphorylated (Nak1/Orb3) forms of Nak1/Orb3 are visible. The extract of the 40 minutes time point was either incubated with -phosphatase (+) or buffer only (–) (right).