CS2ΔSBP1-complemented transgenic cells export PfEMP1 beyond the parasitophorous vacuole. (A) Western blot showing expression of SBP1 in different stages of CS2WT and CS2ΔSBP1-complemented cells. Saponin pellets of erythrocytes infected with ring- (R), trophozoite- (T), and schizont-stage (S) CS2WT and CS2ΔSBP1-complemented parasites were loaded to detect the level of expression of SBP1. For each cell line, samples were taken from the same culture dish at different time points. The blot was stripped and reprobed with HSP70 to show the amount of parasite material loaded. The cross-reactive band at 80 kDa is shown for the same purpose. Saponin pellet of CS2ΔSBP1 trophozoite-infected erythrocytes was loaded as a control. (B) Immunofluorescence assay to assess trafficking of PfEMP1 in erythrocytes infected with CS2ΔSBP1-complemented parasites. Cells were probed with anti-ATS antibody showing typical PfEMP1 localization with protein detectable within the parasite and in prominent punctate structures in the erythrocyte. These structures were shown to colocalize with the Maurer clefts protein SBP1. The amount and timing of SBP1 expression is different in the CS2ΔSBP1-complemented cells because of the episomal expression under a different promoter. DAPI was used to stain the nuclear DNA.