(A) Sml1 is degraded upon colocalization. A wild-type strain was left untreated or was treated 100 ug/ml Zeocin for 4 hr. Strains expressing checkpoint fusions −array and +array were arrested with nocodazole, and galactose was added to induce fusions. The westerns were blotted against endogenous Sml1 and Rad53-HA. A sml1 delete strain served as control for specificity (last lane).
(B–D) Strains containing fusions −array (B), +array (C), and +array, rad9 (D) were arrested in nocodazole, induced with galactose for 1 hr while arrested, and then released into media with α factor. A time course of FACS analysis is shown starting at 2 hr (120′) into galactose induction.
(E) Analysis of Rad53-HA by western blot of samples taken from (B)–(D).