Figure 1.

Comprehensive analysis of in vitro MKK substrate specificity. (A) Combinations of MKK/MPK purified fusion proteins comprising nine wild-type MKK (MKK^WT) and activation loop mutants (MKK^EE) and 10 MPKs, were mixed in 96-well plates with MBP and kinase buffer in the presence of [γ-³²P]-ATP. The proteins were transferred to PVDF membranes and phosphorylated MBP was detected on film. A representative blot is shown. (B) The kinase activity, indicated by phosphorylated MBP, was estimated by measuring the amount of incorporated [γ-³²P]-ATP in all reactions in two or three technical replicates. Spot intensities were obtained from the PhosphorImager exposure. Graphical representation of the likelihood of interaction (−log [P-value]) for the blot in A is shown. The level of significance (0.0143) was represented as the black threshold line at 1.8 U on the Y-axis.