FIG. 3.

Oxidants prolong activation of ERK and perturb the metabolism of c-Fos and c-Jun during cell cycle reentry. Serum-starved C10 cells were stimulated with medium containing 10% FBS, 10% FBS with 1 mM SIN-1, or 10% FBS with SIN-1 and 2,000 U of catalase/ml as indicated. Total-cell lysates were prepared at the indicated times after serum stimulation and analyzed by immunoblotting for the expression of phosphorylated Akt, total Akt, and cyclin D1 (A); phosphorylated ERK1,2, total ERK1,2, and β-actin (B); and total c-Jun and total c-Fos (C). (D and E) Quiescent C10 cells were stimulated with medium containing 10% FBS for 1 h. (D) Total-cell lysates were prepared and treated with CIP (25 U) or CIP and sodium orthovanadate (NaVO₄, 10 mM), and the electrophoretic mobility of c-Fos was examined by immunoblotting. (E) Phosphorylation of c-Fos on threonine 325 was confirmed with an antibody specific for this modification.