FIG. 2.
Expression and enzymatic characterization of TbPI4KIII-β. (A) Immunoblot of Flag-tagged TbPI4KIII-β expressed in COS-7 cells and immunoprecipitated with anti-Flag M2 resin. Anti-Flag antibody detects a band migrating at between 50 and 75 kDa in immunoprecipitates from cells expressing TbPI4KIII-β (lane 2) but not in those from control cells expressing empty vector (lane 1). (B) Kinase assay of the immune complex of Flag-tagged recombinant TbPI4KIII-β. Immunoprecipitated proteins were tested for PtdIns kinase activity in the presence and absence of 100 μM wortmannin (WT), which inhibits mammalian PI4KIII-β activity. Reaction mixtures contained 200 μM PtdIns, 0.3% Triton X-100, 400 μM ATP, and 15 mM MgCl2, and reactions were carried out at room temperature for 30 min. Radioactive PI4P is detected by TLC only in immunoprecipitates expressing TbPI4KIII-β and is prevented from forming by WT. Triplicate measurements are shown.