Fig. 8. Genetic interaction between ROT1 and CLB2. (A) Clb2 protein level in extracts from cultures of the CLB2-HA (JCY285) and the CLB2-HA tetO₇:ROT1 (JCY286) strains grown on YPD and incubated in the presence of 5 µg/ml doxycycline for 8 hours, were examined by western analysis. A control extract from the wild-type strain is included. A loading control of total protein is shown in the lower panel. (B) Protein kinase activity in HA immunoprecipitates from extracts were assayed using histone H1 as a substrate. Lower panels show the Clb2 and Cdc28 protein levels in the immunoprecipitates. A control extract from the wild-type untagged strain is included. (C) Tenfold serial dilutions from exponentially growing cultures of the wild-type (CML240) and tetO₇:ROT1 (JCY216) strains transformed with an empty vector or a plasmid expressing the CLB2 gene under the control of the GAL1 promoter were spotted onto YPD or YPGal medium containing 0.5 µg/ml doxycycline and incubated at 28°C for 3 days. (D) Tenfold serial dilutions from exponentially growing cultures of the wild-type (W303), tetO₇:ROT1 (MCY68), clb2 (CD104-2c) and clb2 tetO₇:ROT1 (JCY255) strains were spotted onto YPD medium with or without 5 µg/ml doxycycline and incubated at 28°C for 3 days. (E) Exponentially growing cultures of the wild-type (W303), tetO₇:ROT1 (MCY68), clb2 (CD104-2c) and clb2 tetO₇:ROT1 (JCY255) strains were incubated in the presence of 5 µg/ml doxycycline for 8 hours. Graph shows the distribution of cells at different cell cycle stages. Values are the mean+s.d. of three experiments. DIC images show DAPI staining of DNA and Alexa Fluor 498-labeled phalloidin staining of F-actin of the clb2 tetO₇:ROT1 cells.