Fig. 10. Control of Clb2 protein stability by Rot1. (A) Wild-type (CML240) and tetO₇:ROT1 (JCY216) cells transformed with a plasmid bearing a GAL:CLB2-HA gene were grown on raffinose (raf), incubated in galactose (gal) for 30 minutes and transferred to glucose medium. Clb2 protein level decay was examined at the indicated times by western analysis. Cdc28 protein level is shown as a loading control. (B) Exponentially growing cultures of the cdc15 (MCY123) and cdc15 tetO₇:ROT1 (MCY151) cells expressing HA-tagged Clb2 and Myc-tagged Sic1 were arrested by incubation at 37°C in the presence of 5 µg/ml doxycycline. After 4 hours (>95% of telophase cells in both cases), cells were transferred to 28°C and Clb2 and Sic1 protein levels were analyzed at the indicated times by western analysis. (C) Wild-type (CML240) and tetO₇:ROT1 (JCY216) cells bearing the GAL:CLB2-HA gene were grown on raffinose and arrested by -factor in the absence or presence of 5 µg/ml doxycycline. After 4 hours (>96% of unbudded cells in all the cases), galactose was added to the arrested cells and, after 30 minutes, protein synthesis was blocked by the addition of cycloheximide (chx). Clb2 protein level decay was examined at the indicated times by western analysis. Cdc28 protein level is shown as a loading control.