Figure 5. SB505124 directly targets TgMAPK1.

A. Strategy for endogenously tagging TgMAPK1 with 3×HA tag. B. Immunoprecipitated TgMAPK1-HA was separated by SDS-PAGE for western blotting antibody and in vitro kinase assays. C. Equivalent volumes of RH WT or RH:TgMAPK1-HA lysate were added anti-HA sepharose beads, then washed and then processed for in vitro autokinase assays. The lysates were then separated by SDS-PAGE and visualized by autoradiography. Shown is a representative assay. D. Equivalent amounts of TgMAPK1-HA was immunoprecipitated from RH:TgMAPK1HA lysates using anti-HA sepharose beads and processed for in vitro kinase assays in the presence of increasing concentrations of SB505124. A representative assay with relative amounts of TgMAPK1 activity in each reaction is shown. E. Dose response curve showing averaged data and standard deviations from 3 experiments. F. Lysates (80 µg) were prepared from RHΔKu80ΔHPT (WT), RHΔKu80ΔHPT:TgMAPK1^WT-HA, and RHΔKu80ΔHPT:TgMAPK1^{ts -HA} parasites grown at 34°C. Epitope-tagged TgMAPK1 was then detected using rat anti-HA antisera in the whole cell lysate and the flow through and immunoprecipitates following immunoprecipitation using rabbit anti-HA antibody conjugated beads. G. Immunoprecipitates of the indicated HA-tagged TgMAPK1 alleles were washed in kinase assay buffer and then incubated with γ³²P-ATP for 60′ at 34°C. Shown are triplicate samples prepared from the same lysates immunoprecipitated in F. The experiment was repeated 3 independent times and representative gels are shown.