Research Paper Volume 8, Issue 4 pp 810—830

Figure 4. Fkh1 instability occurs specifically in mitosis. (A) WT cells expressing endogenous FKH1-TAP were grown to mid log. CHX was added to the asynchronous cells to stop further protein synthesis. Samples were removed every 30 minutes and assessed by Western analyses with the antibodies shown. (B) The experiment described in (A) was performed, except the cells were arrested in G1 using α-factor. (C) The experiment described in (A) was performed, except the cells were arrested in mitosis using nocodazole. (D) The experiment described in (A) was performed, except the cells were arrested in S phase using hydroxyurea. (E) Blots from 3 repeat experiments of (A), (B), and (C), and 2 repeats of (D), were imaged with Fkh1-TAP levels compared over time to visualize Fkh1 stability. Standard error of the mean is shown. Samples were also removed following cell treatment for flow cytometry to verify cell cycle arrest. (F) apc10Δ Fkh1-TAP cells were arrested in G1 using α-factor. CHX was then added with samples removed every 30 minutes for protein analyses.