Figure 4.

TNFα production by H₂O₂ in ES-derived cardiac myocytes. (a) ELISA assay. Wild-type and MEKK1−/− ESCM were treated with 0.1 mM H₂O₂ for 6 h in the presence and absence of drugs. TNFα concentration in the supernatant was measured as described in Materials and Methods. (b) Immunoblot. MEKK1−/− ESCM were treated with 0.1 mM H₂O₂ for 6 h in the presence and absence of drugs. Total cellular proteins were extracted and expression of TNFα in cardiac myocytes was detected by immunoblot analysis by using a specific antibody against TNFα. (c) TNFα promoter reporter assay. pGL3TNF (500 ng) and pRL-TK (50 ng) were cotransfected into wild-type ESCM with 3 μg of either pMEKK1-K1253M, pJNK1-APF, or an empty pCEP4 vector by using a liposome method. Cells were incubated for 24 h and treated with hydrogen peroxide (0.1 mM) for an additional 6 h. Luciferase activities were determined by using a luminometer. TNFα promoter activity was normalized for transfection efficiency based on the cotransfected Renilla luciferase reporter construct.