The activity of Cdc13-des2-HBD is regulated by estradiol. A, Cells transformed with plasmids carrying the nmt.cdc13-des2 and nmt.cdc13-des2-HBD constructs were serially diluted (4X) and plated onto minimal plates with and without thiamine and estradiol as indicated. The cells carrying the ERHBD tagged cdc13-des2 growing poorly in the presence of estradiol are highlighted with a white rectangle. B, Expression of Cdc13-des-HBD results in anaphase delay in the presence of estradiol. The nmt promoter was induced for 20 h before addition of estradiol for 1 h. Anaphase index is shown before and 1 h after addition of estradiol. Bars show anaphase indices in the presence (left panel) and absence (right panel) of thiamine. Anaphase index observed in wild type cells is shown for comparison. The bar representing the tagged construct in the presence of estradiol is shaded. C, Cdc13 levels are not increased by the presence of the tag or estradiol. Cells carrying the nmt81.cdc13-des2 and nmt81.cdc13-des2-HBD plasmids were grown in minimal medium in the presence of thiamine, then thiamine was washed out to induce the nmt promoter. Estradiol was added to half of the cultures after 20 h induction. Samples for protein extracts were taken at the indicated times. TCA extracts were made and western blot analysis was performed using the SP4 anti-Cdc13 antibody  and the anti-PSTAIRE (Santa Cruz) antibody to detect Cdc2 which serves as loading control.
Bøe et al. BMC Cell Biology 2008 9:23 doi:10.1186/1471-2121-9-23