The immunofluorescence assays (IFAs) were performed as described previously [49 (link)]. Briefly, tachyzoite-infected DF-1 cells were fixed with 4% paraformaldehyde. After three washes with PBS, the cell membranes were permeabilized and the samples were blocked by incubation with 0.25% Triton X-100/PBS and 3% bovine serum albumin for 30 min at room temperature. The primary antibodies used were mouse anti-HA (1:500; Sigma Aldrich, St. Louis, MO, USA), rabbit anti-GAP45 (1:250), rabbit anti-SAG1 (1:150), and rabbit anti-IMC1 (1:200). TgActin, TgSAG1, and TgIMC1 were prepared by our laboratory. The location of the primary antibody was visualized by using a mixture of FITC-conjugated goat anti-mouse IgG [H+L] (1:75) and Cy3-conjugated goat anti-rabbit IgG [H+L] (1:250). The nuclear material was co-stained with 4′,6-diamidino-2-phenylindole (DAPI).
For the immunoblotting assays, 107 parasites were collected and purified by filtration through a 5 µm filter membrane and lysed with RIPA buffer (Solarbio, Beijing, China). The primary antibodies used were mouse anti-GFP (1:6000; Abways, Shanghai, China), mouse anti-HA (1:8000; Sigma Aldrich, St. Louis, MO, USA), and mouse anti-Actin (1:500). Horseradish peroxidase-conjugated antibodies were used as secondary antibodies (1:5000; CWBIO, Beijing, China).
For the immunoblotting assays, 107 parasites were collected and purified by filtration through a 5 µm filter membrane and lysed with RIPA buffer (Solarbio, Beijing, China). The primary antibodies used were mouse anti-GFP (1:6000; Abways, Shanghai, China), mouse anti-HA (1:8000; Sigma Aldrich, St. Louis, MO, USA), and mouse anti-Actin (1:500). Horseradish peroxidase-conjugated antibodies were used as secondary antibodies (1:5000; CWBIO, Beijing, China).