Fig. 3. Chk1 protein accumulates in the nucleus after DNA damage. (A) A strain with integrated Chk1:HA was examined by immunofluorescence with anti-HA antibody. Cultures grown to mid-log phase were treated with 40 µM CPT for 2.5 hours to generate DNA damage, then fixed and processed for immunofluorescence. Antibody to a cytoplasmic protein Ded1 is also shown. Bar, 10 µm. (B) Quantification of immunofluorescence signal was performed by measuring pixel intensity in the nuclei and in an equivalent area of the cytoplasm. Nuclear to cytoplasmic ratios of these signals were then generated and cells were sorted into the three categories shown. (C) Quantification was performed and is presented for the integrated wild-type chk1:HA allele (an average of two independent experiments is shown). (D) Quantification is presented on a similar immunofluorescence experiment performed with a strain deleted for endogenous chk1 and instead expressing Chk1:HA from the pSP1 plasmid (an average of four independent experiments is shown).