FIG. 7.

Addition of osmostabilizing agents or loss of PLC1 activity stabilize Ume3p in response to oxidative stress. (A) Ethanol-shock-induced Ume3p degradation was monitored in wild-type cultures (RSY10) grown in SD medium (wild type) in the presence of 10% sorbitol or in hog1 (W303 hog1Δ) or plc1 (JTY2304) mutants as indicated. Samples were taken prior to (0 min) and at various times after treatment as indicated. Protein extracts were prepared, and Ume3p levels were determined by Western blot analysis. The vector lane controls for nonspecific cross-hybridization of the myc monoclonal antibody used to detect the epitope-tagged Ume3p derivative. Tub1p levels served as protein loading controls. (B) Oxidative stress. The experiments described in panel A were repeated, except that 0.4 mM H₂O₂ was added to the cultures to generate oxidative stress. Samples were taken at the indicated times.