Figure_5.tif (2.57 MB)
Download file

SB505124 directly targets TgMAPK1.

Download (0 kB)
posted on 12.06.2014, 03:20 authored by Kevin M. Brown, Elena Suvorova, Andrew Farrell, Aaron McLain, Ashley Dittmar, Graham B. Wiley, Gabor Marth, Patrick M. Gaffney, Marc Jan Gubbels, Michael White, Ira J. Blader

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:TgMAPK1WT-HA, and RHΔKu80ΔHPT:TgMAPK1ts-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.