FIG. 9.

Identification of downstream targets of MKK4. (A) Total RNAs were extracted from control (ctrl) and mutant (mt) E14 embryonic forebrains, and the amounts of various transcripts were measured by quantitative real-time PCR. The data are expressed relative (n-fold) to the mRNA extracted from the control brain and correspond to the means and standard errors of three independent experiments performed in triplicate. P values relative to the control sample are indicated by asterisks: ***, P < 0.001; **, P < 0.01; *, P > 0.05. (B) Phosphorylation of MAP1B and NF-H was examined by immunoblot analysis of brain extracts (20 μg) using antibodies against the phosphorylated forms of MAP1B and NF-H (SMI31). Absence of MKK4 was correlated with a specific defect in MAP1B and NF-H phosphorylation. Pan-antibodies to MAP1B (N19) and NF-H (N52) showed no significant difference in the expression levels of the proteins between the two genotypes. Note that the detection of phospho-NF-H (P-NF-H) by SMI31 in control extracts was correlated with the retarded mobility of the protein after SDS-PAGE. Similar results were obtained in two independent experiments. (C) Consistent with the defective phosphorylation of NF-H in the mutant extracts, cerebellar and cerebral cortices lacking MKK4 displayed strong immunoreactivity against SMI32, an antibody that specifically reacts with the nonphosphorylated form of NF-H. No difference was observed in the immunostaining of the control and mutant cortices with the anti-MAP2 antibody. Scale bar, 50 μm.