Figure 2. APC function is required for normal Est1p degradation during G1 phase.

(A) HA₃-Est1p stability increases when APC function is compromised. Western blots of Est1p stability assays from strain K4438 (cdc16-123) harboring pKF600 (GAL1-HA₃-EST1) plus either a complementing vector pRS416-CDC16 (labeled “CDC16”) or an empty vector pRS416 (labeled “cdc16-123”) were conducted as described in Materials and Methods. An uninduced sample (Raff) served as a negative control for HA₃-Est1p specificity. (B) HA₃-Est1p is stabilized in APC deletion mutants. Western blots of Est1p stability assays from strains YKF802 (Wild Type), YKF803 (apc9Δ), YKF804 (mnd2Δ), YKF805 (swm1Δ), YKF806 (clb2Δ) and YKF807 (clb2Δcdh1Δ) containing pVL242RtoA (P_GAL1-HA₃-EST1) were conducted as described in Materials and Methods. For YKF805 (swm1Δ), an uninduced asynchronous sample (Raff) served as a positive control for Clb2p detection and negative control for HA₃-Est1p specificity, while an uninduced asynchronous sample of strain YKF806 (clb2Δ) served as a negative control for Clb2p detection. (C) Quantification of results shown in (A). Bars represent the average HA₃-Est1p half-life from three independent biological replicates. Error bars are standard deviation of the mean (p-value = 0.08 by two-tailed t test). (D) Quantification of results shown in (B). Bars represent the average HA₃-Est1p half-life from independent biological replicates: n = 3 for all strains except clb2Δcdh1Δ, where n = 4. Error bars are standard deviation from the mean. By two-tailed paired t-test, there is a significant difference between the control (WT) and swm1Δ (p-value 0.0002) but not between WT and apc9Δ (p-value 0.49) or mnd2Δ (p-value 0.83). There is a significant difference between the control (clb2Δ) and clb2Δcdh1Δ strains (p-value 0.003). Significant differences are denoted by *.