(A-B) Tf-LPA vesicle localisation was determined using indicated parasite strains expressing markers for the parasite trafficking system: dd-RAB18-myc: ER, dd-RAB4-myc: Golgi, VPS53-HA: TGN, VPS35-HA: Retromer/ELC, CHC-HA: Golgi, TGN, AP2α-HA parasites [31,32,43,44]. Antibodies against the VAC (α-CPL [37]), rhoptries (α-ROP1), micronemes (α-MIC2), and dense granules (α-GRA1) were used after fixation on RH parasites. (A) Representative pictures of colocalisations are shown (see also S4 Fig); scale bar, 1 μm. (B) Quantification of colocalisations. Mean values of three independent assays are shown ± SD. (C) Accumulation of Tf-LPA in the VAC over time. Percentage of colocalisation between Tf-LPA and CPL was determined from 0 to 30 minutes after Tf-LPA addition. Mean values of three independent assays are shown ± SD. For each bar graph, the corresponding data can be found in S1 Data. CHC, clathrin heavy chain; CPL, cathepsin L; dd, destabilisation domain; ELC, endosome-like compartment; ER, endoplasmic reticulum; Tf-LPA, Top-Fluor lysophosphatidic acid; TGN, Trans-Golgi network; VAC, vacuolar-like compartment; VPS, vacuolar protein sorting-associated protein.