FIG. 2.

The disruption of the mkk4 gene significantly reduces JNK activity. (A) Tissue extracts prepared from embryos and mice at various ages that were homozygous for the mkk4-flox allele and expressed (mutant [mt]) or did not express (control [ctrl]) Cre were analyzed for ΜΚΚ4, MKK7, JNK, and p38 MAPK expression by immunoblot analysis using specific polyclonal antibodies. The detection of tubulin expression was performed to monitor protein loading. (B) The inactivation of the mkk4 gene in the brains of the mutant mice was confirmed by immunostaining of sagittal sections of the cerebral cortex, the hippocampus, and the cerebellum at P6 with an antibody to MKK4. (C) Endogenous JNK and p38 MAPK activities were measured by protein kinase assay (KA) in the presence of [γ-³²P]ATP. The radioactivity incorporated into GST-cJun or GST-ATF2 was quantitated after SDS/PAGE by phosphorimager analysis. The data correspond to the means and standard errors of three independent experiments. (D) Sagittal sections of different areas of control and mutant brains at P3 or P6 were immunostained with antibodies to phospho-c-Jun (Ser63) and phospho-ATF2 (Thr71). The results demonstrate that the loss of MKK4 prevents the phosphorylation of c-Jun and ATF2 in the nervous system.