Immunofluorescence assays (IFA)

All IFAs were carried out using the similar following protocol. Intracellular parasites were fixated using 4% PFA for 30 min. The coverslips were incubated with primary antibodies and then secondary antibodies coupled to Alexa Fluor-488 or to Alexa-Fluor-594. Primary antibodies used for IFAs include: anti-TgEno2, anti-TgISP1³⁴, anti-TgIMC1 (a gift from Pr. Ward, U. Vermont), anti-TgCentrin1 (a gift from Pr. Gubbels, Boston College), anti-TgChromo1²², anti-TgSortilin (Golgi), anti-TgACP (Plastid), anti-TgTom40⁴¹ (mitochondrion), anti-HA (Sigma-Aldrich) and anti-myc (abcam) antibodies were used at the following dilutions: 1:1000, 1:500, 1:500, 1:500, 1:500, 1:500, 1:500, 1:500, 1:200, respectively. Confocal imaging was performed with a ZEISS LSM880 Confocal Microscope or Apotome Microscope at 63 magnification. All images were processed using Carl Zeiss ZEN software. Quantification of immunofluorescence assays was carried out manually by counting the concerned signal corresponding to the organelle by visual observation. Signal corresponding to 100 parasites was counted for each replicate.