Fig. 1. Both MKK4 and MKK7 mediate H₂O₂-induced SAPK activation and cell death. (A) U937 cells were exposed to 1 mM H₂O₂ for the indicated periods. The cells were lysed, and the SAPK, ERK and p38 MAPK phosphorylation levels were analyzed by western blotting using antibodies specific to the phosphorylated forms of each MAPK. The protein levels of these MAPKs were also probed using their specific antibodies. (B) The SAPK, ERK, p38 MAPK, MKK4 and MKK7 activities in the lysates were analyzed by in vitro kinase assay. The substrates were recombinant c-Jun protein for SAPK, PHAS-1 for ERK and p38 MAPK, and SAPK protein for MKK4 and MKK7. (C) The U937 cells were stably transfected with the pcDNA vector containing the dominant negative mutant of either MKK4 or MKK7. These transfectants and the cells that received the empty control vector were exposed to 1 mM H₂O₂ for 30 minutes. Levels of SAPK activity in the treated and untreated control cells were compared by in vitro kinase assay. (D) The transfectants were treated with the indicated H₂O₂ concentrations for 48 hours and the cellular viability was analyzed by flow cytometry.