FIG. 7.

Ectopic expression of phosphatase-inactive CDC25A(C430S) suppresses activation of the stress kinase cascades upon oxidative stress. (A) The expression of CDC25A(C430S) after a 4-h induction with doxycycline (Dox; 10 ng/ml) in 293 cells carrying a tetracycline-inducible transgene. The sum of expression of CDC25A(C430S) and endogenous CDC25A was measured by immunoblotting. (B) Ectopic CDC25A(C430S) expression diminishes the activation of ASK1, JNK1 and p38 by H₂O₂ treatment. Cells were treated with doxycycline for 4 h and then exposed to H₂O₂ (2 mM) for 20 min. For the activities of the stress-responsive kinases, cell extracts were immunoprecipitated with antibodies against ASK1, JNK1, or p38, and the activities were assayed as described in Materials and Methods. Phosphorylation of MKK3/6 was examined by Western blotting with an antibody specific for phosphorylated active forms. Total expression of MKK3, JNK1, and p38 was determined by Western blotting.