Figure 4.

Cdc45 Directly Enhances Rad53-Dependent Sld3 Phosphorylation In Vitro and Is Phosphorylated by Rad53 In Vivo

(A) Kinase assay using Rad53, Sld3, and either BSA or Cdc45. Sld3, Cdc45, and BSA were in 25-fold excess over Rad53. Top: Coomassie-stained gel. Middle: autoradiogram. Bottom: Rad53 western blot. Asterisks mark degradation products of Sld3.

(B) Quantitation of the kinase assay in (A). Error bars represent SD; n = 3.

(C) As in (A), except using either wild-type Cdc45 or the Cdc45-2A mutant.

(D) As in (B).

(E) Western blot of the indicated strains released from alpha factor (time 0) into 200 mM HU. The Cdc45 western blot was performed using PhosTag PAGE. 60+λ is the addition of λ phosphatase to the 60-min HU time point.

(F) As in (E).