Figure 3. Cell-cycle oscillation of Est1p requires Cdh1p.

(A) Est1 protein levels oscillate through the cell cycle. Strain YKF808 (cdc15-2 EST1-MYC₁₃) was grown asynchronously at 23°C to mid-log phase and shifted to the restrictive temperature (37°C) for 3.5 hrs. When 95% of the cells were arrested, as monitored by bud-index (Figure S2), the culture was returned to the permissive temperature (23°C; time 0). Whole-cell extract was prepared from samples harvested every 20 mins following release and western blotted using anti-MYC, anti-Clb2p, and anti-Actin antibodies, as indicated. YCM191 (cdc15-2) served as the untagged (No MYC) control for Est1-MYC₁₃p and was harvested following the 37°C incubation period. Est1-MYC₁₃p and Clb2p intensity at each time were normalized to input (actin) and starting amount (time 0). Bars represent the average of four independent biological replicates for Est1-MYC₁₃p (light) and Clb2p (dark); error bars are standard deviation of the mean. (B) Deletion of CDH1 perturbs the oscillation of Est1-MYC₁₃p through the cell cycle. Strain YKF809 (cdc15-2 cdh1Δ EST1-MYC₁₃) was treated as in (A), except the bars represent the average of three independent biological replicates.