PfPI3K, a PI3 kinase from P falciparum. (A) Schematic diagram showing domain architecture of PfPI3K. The helical and catalytic domain of PfPI3K is separated by a linker. In addition, a C2 domain is present near its N-terminal end. (B) Equal amounts of cell lysates prepared either from either uninfected erythrocytes (E left panel) or trophozoite stage parasites (Pf right panel) were electrophoresed on 7% SDS-polyacrylamide gel electrophoresis gel, and Western blot analyses were performed using anti-PfPI3K antisera on both erythrocyte as well as the parasite lysate. A control Western blot with preimmune antisera was performed to probe the parasite lysate (middle panel). (C) PfPI3K was immunoprecipitated from trophozoite lysates, and PfPI3K-IP was used in a lipid kinase assay wherein either PI or PI3P (left panel), PI4P, or PI(4,5)P2 (right panel) was used as substrate. Phospholipids were separated on a TLC along with phosphoinositide standards, and radiolabeled lipid products were detected using a phosphorimager. (D) The activity of PfPI3K-IP from trophozoite lysates was assayed using PI4P as substrate (as described in panel C) in the presence of 2.5μM wortmannin (left panel), or 50μM LY294002 (right panel) or DMSO (−).