Figure 9.

The mutant IAK1^D287N shows reduced kinase activity. Cell extracts from NIH 3T3 cells transiently expressing the flag-tagged IAK1 and IAK1^D287N constructs were prepared, and the wild-type and the mutant form of the kinase was selectively isolated using the anti–flag M2 affinity gel as described in the Materials and Methods section. Mock-transfected cells were used as the control. The affinity-purified wild-type and mutant form of the IAK1 kinase was used for the in vitro kinase activity using myelin basic protein as the substrate. The experiment was repeated three times, and the typical result from a single experiment is presented here. The incorporation of [γ-³²P]ATP into myelin basic protein was measured using a Molecular Dynamics phosphoimager, and the result is presented in the form of a bar diagram after background correction. The inset shows the autoradiographic representation of the same result. Lane 1, control mock-transfected cells; lane 2, IAK1; lane 3, IAK1^D287N.