Fig. 1. Identification of residues in Chk1 required for the interaction with Rad24. (A) Domain structure of Chk1 highlighting the region required for interaction between Chk1 and Rad24 (amino acids 286-319). (B) Result of yeast two-hybrid ß-galactosidase assay. Strains transformed with the pACT/Rad24 plasmid and the indicated alleles of chk1 in the pAS2/Chk1 plasmid were exposed to X-Gal. Only the wild-type allele and the kinase domain mutant K38A show expression of the lacZ gene as revealed by X-gal reactivity. (C) Western blot of lysates from cells expressing the indicated alleles of Chk1 that had (+) or had not (–) been exposed to the topoisomerase-I poison camptothecin (CPT). Chk1 is detected with anti-HA antibody to detect the tag on the C-terminus of Chk1. Only the wild-type protein undergoes the mobility shift representative of Chk1 phosphorylation. (D) Immunoprecipitation of lysates shown in C to evaluate the interaction of alleles of Chk1 with Rad24. Immunoprecipitations were carried out with antibodies against Rad24 or with a non-immune antibody. The immunoprecipitates were separated by SDS-PAGE, transferred to nitrocellulose and probed by immunoblot with antibody against the HA tag on the C-terminus of Chk1.