Figure 6.

Specific catalytic activity of endogenous MKK4 in mouse macrophages in response to TNFα. (A Upper) Immunoprecipitation of endogenous MKK4 followed by an in vitro kinase assay using kinase-inactive p46 JNK/SAPK showed that MKK4 is activated in a time-dependent fashion. (Lower) Immunoblot with the MKK4 (C-20) antibody showed equal loading of sample. (B Upper) Immunoprecipitation of MKK4 followed by an in vitro kinase assay with the kinase-inactive p54 SAPKβ-GST substrate revealed that MKK4 is able to phosphorylate p54 JNK/SAPK. (Lower) Immunoblot with the MKK4 (C-20) antibody. The no-substrate lane comprises macrophages stimulated with TNFα (40 ng/ml) for 10 min. These results are representative of three independent experiments.