Figure 2.

CLB2 mRNA levels and Clb2p-Cdc28p activity decrease in response to hypertonic shock. (A) Wild-type MT588 cells were grown to log phase and stressed by the addition of NaCl to 0.4 M. Samples were taken at the indicated times. Total RNA was isolated and probed for CLB2. The same membrane was reprobed for ACT1 as a loading control. (B) Clb2p-Cdc28p kinase activity was assessed by in vitro kinase assays by using histone H1 (HH1) as a substrate. Kinase assays were performed on immunoprecipitated Clb2p-HA Cdc28p complexes isolated from wild-type MT588 cells treated the same as in A. (C) Clb2-HA and Cdc28p levels in total cell lysate used for B were determined by Western blot analysis. (D) Stability of Clb2–HA/Cdc28p complexes was determined by immunoprecipitating Clb2-HAp from the samples used for B and C. Western blots were then probed with anti-Cdc28 and anti-HA antibodies. The band marked with ∗ is IgG. W303 cells lacking the HA tag on Clb2p were used as a control. Similar results were obtained in at least three separate experiments.