Figure 4.

Hypertonic stress induces the phosphorylation of Cdc28p in a Swe1p-dependent manner. Asynchronous cultures of MT588 were stressed by the addition of 0.4 M NaCl. (A) Cdc28p was precipitated with p13^Suc1-agarose beads and analyzed by Western blot. Anti-phospho-Cdc2 was used to determine the phosphorylation state of Cdc28p (see MATERIALS AND METHODS). Membranes were stripped and reprobed with anti-Cdc28. A GAL: wee1⁺ mih1Δ strain and a Cdc28^Y19F-HA strain were used as positive and negative controls, respectively. The decrease in mobility of the Y19F control is the result of the HA epitope tag. (B) Phosphorylation of Clb2-Hap-associated Cdc28p was examined by immunoprecipitating Clb2–HAp/Cdc28p complexes with anti-HA antibodies and protein A/G agarose beads. As a positive control, hyperphosphorylated Cdc28p from GAL: wee1⁺ mih1Δ cells was coprecipitated with p13^Suc1-agarose beads. Western blots were probed with anti-phospho-Cdc2 antibodies and then stripped and reprobed with anti-Cdc28 and anti-HA antibodies. Results were consistent in three separate experiments.