Supplemental Material for Eisenstatt et al., 2020

Figure S1. Endogenous Flag-Cse4 is not mislocalized in cdc7-7 strains. Chromatin lysate from wild type (YMB10043) and cdc7-7 (YMB10040) strains with endogenous Cse4 tagged with Flag was immunoprecipitated against anti-Flag agarose. Input and IP samples were prepared for paired-end ChIP-seq and reads were aligned to the S. cerevisiae S288C reference sequence. A-G. Genome browser of input and ChIP samples for Chromosomes I, II, III, V, X, XI, and XV from wild type (top) and cdc7-7 (bottom) strains with Flag-Cse4. The CEN regions are indicated.

Figure S2. Overexpressed Flag-Cse4 is mislocalized in cdc7-7 strains. Chromatin lysate from wild type (YMB10044) and cdc7-7 (YMB10041) strains overexpressing Cse4 tagged with Flag was immunoprecipitated against anti-Flag agarose. Input and IP samples were prepared for paired-end ChIP-seq and reads were aligned to the S. cerevisiae S288C reference sequence. A-E. Genome browser of input and ChIP samples for Chromosomes II, III, X, XI, and XV from wild type (top) and cdc7-7 (bottom) strains with Flag-Cse4. The CEN regions are indicated.

Figure S3. Endogenous Flag-Cse4 is not stabilized in a cdc7-7 strain. Western blot analysis of protein extracts from wild type (YMB10043) and cdc7-7 (YMB10040) strains with Flag-Cse4 expressed from its own promoter were grown to logarithmic phase of growth. Cells were treated with cycloheximide (CHX, 50 µg/ml) and aliquots were taken at the indicated timepoints. Protein extracts were analyzed using Western blot analysis and blots were probed with anti-FLAG (Cse4) and anti-Tub2. (loading control). The graph shows the quantification of the levels of FLAG-Cse4 remaining after treatment with CHX relative to Tub2 from three independent experiments. Error bars represent SEM.