Figure 5.
Effect of Mkk4 and Mkk7 gene disruption on TNF-stimulated JNK activation and necrosis. (A) Wild-type (WT), Mkk4-/-, Mkk7-/-, and Mkk4-/- Mkk7-/- fibroblasts were incubated (10 min) without and with 10 ng/mL TNF. The amount of tubulin, JNK, and phospho-JNK was examined by immunoblot analysis. The data shown are representative of three independent experiments. (B) Wild-type (WT) fibroblasts were incubated with 10 ng/mL TNF. The amount of tubulin, MKK4, phospho-MKK4, MKK7, and phospho-MKK7 was examined by immunoblot analysis. The data shown are representative of three independent experiments. (C) Wild-type (WT), Mkk4-/-, Mkk7-/-, and Mkk4-/- Mkk7-/- fibroblasts that express ΔN-IκBα were incubated without and with 10 ng/mL TNF. Cell survival after incubation for 6 and 24 h was examined by staining with crystal violet. The data shown are the mean ± SD of triplicate determinations. The data are representative of three independent experiments. (D) Wild-type (WT), Mkk4-/-, Mkk7-/-, and Mkk4-/- Mkk7-/- fibroblasts were incubated with 10 ng/mL TNF. The effect of ΔN-IκBα expression was examined. The amount of tubulin, ΔN-IκBα, and JNK was examined by immunoblot analysis. JNK activity was examined in an in vitro kinase assay (KA) using the substrate c-Jun. The data shown are representative of three independent experiments.