Figure 4.

Regulation of protein levels of individual MBF factors Cdc10, Res1p, Res2p, and Rep2p upon HU treatment. (A–C) Cells treated with 8 mM HU were sampled at various time points as indicated for Western blot analysis. Strains bearing a sole copy of cdc10⁺-3HA, res1⁺-3HA, and res2⁺-3HA were used for analysis of protein levels after HU treatment in A, and strains bearing a sole copy of rep2⁺-3HA were used as indicated in B. The tubulin levels were used as loading control. Membranes in B were stripped and reblotted for determination of Cig2p levels. Individual bands were quantified using ImageJ software (National Institutes of Health, Bethesda, MD). Fold changes of Rep2p and Cig2p levels are plotted after 0-h normalization. (D) Average expression profiles of the MCB-cluster in cig2Δ cells. The x- and y-axis indicate expression ratios and time points after HU treatment, respectively. (E) Plating assays for HU susceptibility. Approximately 5 μl of 10-fold series diluted samples were inoculated onto plates supplemented with 5 mM HU and grown at 30°C for ∼2–3 d. Arrows and arrowhead indicate strains with and without noticeable sensitivities to HU, respectively. (F) Plating assays for sensitivity to HU. ∼5 μl of 10-fold series diluted samples were inoculated onto plates containing 2.5 mM HU and supplemented with thiamine (repressed for rep2⁺) or without thiamine (induced for rep2⁺). Arrows indicate that phenotypes of rad3Δ and cds1Δ strains are alleviated when rep2⁺ is over produced.