Research Paper Volume 8, Issue 4 pp 810—830

Figure 2. Fkh1 stability depends on the APC and the proteasome. (A) WT and apc5^CA cells expressing Fkh1-TAP were grown to mid log phase. WT cells were also treated with α-factor to arrest cells in G1. Whole cell lysates were prepared and analyzed by Western analyses with the antibodies shown. (B) The experiment in (A) was repeated 3 times, with all protein bands quantified, normalized to Ponceau S stained gels and plotted. Standard error is shown. * - p≤0.001; ** - p≤0.005. (C) The cells shown were grown to mid log phase, after which total RNA was extracted and used for quantitative reverse transcriptase PCR using primers against FKH1 and ACT1 as a normalization control. (D) The experiment in (C) was repeated 3 times with bands quantified, normalized to ACT1, and plotted. Standard error is shown. (E) WT and rpn10Δ cells expressing endogenous FKH1-TAP were grown to mid log phase, with proteins analyzed by Westerns using antibodies against TAP and GAPDH, with the Ponceau S stained gel confirming equivalency of load. (F) Asynchronous FHK1-TAP cells were incubated with 75 μM MG132 for 3 hours at 30°C prior to preparing cell lysates to inhibit the proteasome. Westerns were then performed using antibodies against TAP and Clb2. (G) The bands from (F) were quantified, normalized to the Ponceau S stained gel and plotted. * - p≤0.05. (H) Cells in (F) were used for flow cytometry to determine cell cycle distribution. (I) Asynchronous FKH1-TAP expressing cells, were treated +/− MG132 as in (F). Cycloheximide (CHX) was then added to inhibit protein synthesis with samples taken every 20 minutes to determine Fkh1-TAP stability.