Fig. 2

TgANT localizes to the IMM. a The PK protection assay was used to determine the accessibility of TgANT to 0.1 mg/ml PK in the presence or absence of the detergent 0.5% TX. After permeabilization by TX, PK could easily pass through organelle membranes to the stroma and digest stroma proteins nonspecifically. Actin was used as a cytoplasmic protein marker, and PK did not need the assistance of TX permeabilization solution for its nonspecific degradation process. CytC is a marker of mitochondrial intermembrane space, and its degradation by PK requires the synergistic effect of the TX permeabilizer. b The TgANT-FLAG-TOM40-HA strain is permeabilized with 0.005% or 0.01% digitonin. TOM40 was used as the markers of the mitochondrial outer membrane. Using 0.005% digitonin only allowed the detection of TOM40, while using 0.01% digitonin, both TOM40 and TgANT were detected, suggesting that TgANT is associated with the IMM. Parasites were labeled with mouse anti-FLAG (green), rabbit anti-HA (red) and DAPI (blue). Scale bar: 2 μm. Abbreviations: CytC, cytochrome c; IMM, inner mitochondrial membrane; PK, proteinase K; TX, Triton-X-100