Hemoglobin accumulation in malaria parasites as result of PfPI3K inhibition. (A) Late ring stage parasites were incubated with DMSO (control), wortmannin, or LY294002 for approximately 5 hours. After releasing the parasite from the erythrocytes, parasite lysates were prepared and Western blotting performed using antihemoglobin antiserum or anti-actin antibody. (i) A representative Western blot from each experiment is shown. Actin was used as a loading control. (ii) The hemoglobin band intensity from Western blots performed on 3 independent experiments in which the parasite was treated with 10μM wortmannin or LY294002 was quantified by densitometry using Kodak 1D image analysis software and is represented by bar graphs. The intensity levels were normalized to the control set at 100, and comparisons were made. The error bars represent SEM. (B) Erythrocytes were preloaded with HRP, infected with parasites, and treated with 10μM wortmannin for 14 hours. After releasing parasites from infected erythrocytes by saponin treatment, HRP levels associated with parasites were determined with a colorimetric enzyme assay. Absorbance values were normalized to the solvent controls set at 100. Error bars represent SEM.