Figure 3

Topology of small VSA at the host cell membrane. a, b Western Blot analysis after protease treatment. MACS enriched infected erythrocytes (mostly schizonts) of the 3D7 strain were left intact (intact), subjected to hypotonic lysis (HL) or permeabilized with saponin (Sap) and subsequently also either treated with trypsin (+) or mock-treated with PBS (−). All samples were solubilized in SDS sample buffer, separated by SDS-PAGE and analysed by immunoblotting. Equivalents of 1 × 10⁷ cells were loaded in each lane. The blots were probed with α-VSA sera as indicated (a). As controls, α-ATS antibodies against the acidic terminal segment of PfEMP1 proteins, α-Spectrin serum against the erythrocyte cytoskeleton protein spectrin and α-SBP1-NT antibodies directed against the N-terminal domain of the MC resident skeleton binding protein SBP1 as well as α-MSP1, α-SERP and α-PP5 were used (b). c Quantification of the small VSA-specific immunoblot signals after surface trypsinization of infected erythrocytes by densitometry. Three replicate experiments were quantified and data are presented as relative density from trypsin treated versus PBS treated samples adjusted to SBP1 (red line). The control proteins spectrin, SERP and PP5 are shown in grey as a reference. T test, **p < 0.01, *p < 0.05.