Intracellular localization of PfPI3K and its trafficking to the host. (A) IFA was performed for localizing PfPI3K, FCP (i) and VARC (ii). FCP (red) is located inside the food vacuole of mature trophozoites. PfPI3K (green) exhibits “vesicular” staining on PVM/PM (black arrows) and the host erythrocyte (white arrows) and in the food vacuole, which can be identified by the presence of black hemozoin. (ii) PfPI3K colocalizes with VARC at the erythrocyte surface. (B) Parasites were treated either with 5μg/mL BFA or DMSO, and immunofluorescence was performed using anti-PfPI3K antisera. BFA treatment blocked the transport of PfPI3K to the erythrocyte. (C) Trophozoite stage parasites were treated with either streptolysin (SLO) or saponin (SAP). The soluble (S) and pellet (P) fractions were used for Western blot (bottom panel) and immunoprecipitation of PfPI3K. PfPI3K-IP was assayed for activity using PI4P as the substrate; the radiolabeled product PI(3,4)P2 was detected by phosphorimaging of TLC plates. Anti-PfHSP70 was used for a control Western blot.