H₂O₂ stimulates G1/S cell cycle transition that is associated with down-regulation of p27 and activation of cdk2. (A) Cells expressing WT and A- or E-Bcl2 mutants were deprived of IL-3 for 24 hours. Cell cycle status was assessed following addition of low concentration of H₂O₂ (ie, 50 μM) in the absence of IL-3 for various times as indicated. (B) Cells expressing WT Bcl2 were treated with various concentrations of H₂O₂ for 30 minutes. The cells were harvested, washed, and lysed in detergent buffer. Western blot analysis was performed to detect phosphorylated ERK1/2 (p-ERKs) or total ERK1 and ERK2 proteins by using a phosphospecific ERK antibody or a mixture of ERK1 and ERK2 antibodies, respectively. (C) Cells were treated with various concentrations of H₂O₂ for 30 minutes. Cdk2 was immunoprecipitated from cell lysates and incubated with purified histone-1 in an in vitro kinase assay as described in “Materials and methods.” Reaction mixtures were subjected to SDS–PAGE. Cdk2 activity was analyzed by autoradiography. Cdk2 protein was determined by Western blotting using Cdk2 antibody. (D) Cells were treated with various concentrations of H₂O₂ as described in (C). Cells were harvested and lysed in detergent buffer. Expression levels of p21 and p27 were analyzed by Western blotting using p21 and p27 antibodies. The data represent the mean ± SD of 3 determinations.