Figure 9. Mutation of lysine 40 or aspartic acid 136 or double mutations abolishe HIPK4 kinase activity.

Human 293 kidney cells were transiently transfected with expression vectors carrying wild type or mutant S-tagged HIPK4. Cells were also transfected with same vector without HIPK4 insert. Cell lysates were prepared for S-tag protein pull-down and the pull-down precipitates were subjected to in vitro kinase assay using [γ-³²P]-ATP and MBP as a substrate. Signals indicating phosphorylated proteins were detected by autoradiography.