3608 Nucleic Acids Research, 2009, Vol. 37, No. 11 Figure 4. Cdk1 and Tel1 phosphorylate Cdc13 at late S and G2 phases. (A) After a factor arrest and release, lysates from wild-type, cdc13-T308A and tel1 cells were collected at 20-min intervals for 180 min and Cdc13 was analyzed by western blot analysis. Data of FACS analysis are shown at the bottom. Hypo- and hyper-phosphorylated Cdc13 are marked with single and double asterisks, respectively. (B) The FACS data of wild-type and cdc13-T308A at 40 min are shown. Percentages of cell cycle stages were quantiﬁed according to the manufactures instructions (Becton Dickinson). (C) Lack of checkpoint activation in cdc13-T308A cells. Lysates of wild-type and cdc13-T308A cells were collected and Rad53 phosphorylation was analyzed by western blot analysis using a Rad53 antibody. Bleomycin treatment (5 mU/ml, 3 h) was used as a positive control. Figure 5. Telomere analysis of cdc13 mutants. Cdk1 phosphorylation sites of Cdc13 were mutated from T or S to A, D or E, and mutations were created in wild-type, hdf1, mec1 and tel1 strains. DNA from each strain was digested with XhoI, separated in a 1% agarose gel, transferred to a nylon membrane and hybridized with a Y0 probe. Cells used in this experiment had pop-out the YIP cdc13 URA3 plasmid for at least 50 generations.