Horseradish peroxidase conjugated antibodies

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).

pCMV-Flag-RTN and pCMV-Flag-β-Tubulin were transfected in HEK293T. The GST-fusion protein of TgMORN2 was recombinantly expressed in E. coli BL21. The cells were pelletized and lysed by sonication in protein extraction buffer. After centrifugation at 10,000× g for 30 min, the bacterial lysates were coupled to glutathione sepharose beads (Beyotime, Shanghai, China) for 4 h. The beads were washed 3 times with washing buffer and subsequently incubated with HEK293T cell lysates for 4 h. After 12% SDS-PAGE, the proteins were transferred onto PVDF membranes using the Western blot method. The primary antibodies used were rabbit or mouse anti-GST (1:8000; Proteintech, Wuhan, China) and rabbit or mouse anti-Flag (1:8000; Abways, Shanghai, China). Horseradish peroxidase-conjugated antibodies were used as secondary antibodies (1:5000; CWBIO, Beijing, China).

HEK293T cells co-transfected with the corresponding plasmids were lysed in 400 µL of RIPA buffer containing protease inhibitors and immunoprecipitated with protein A+G (Beyotime, Shanghai, China). The experiment was continued according to the manufacturer’s instructions by first adding 1 μL of mouse IgG anti-Flag to the lysate and then adding it to the protein A+G. The bound proteins were eluted, and the samples were subjected to Western blotting. The primary antibodies used were mouse anti-GFP (1:6000; Abways, Shanghai, China) and mouse anti-Flag (1:8000; Abways, Shanghai, China). Horseradish peroxidase-conjugated antibodies were used as secondary antibodies (1:5000; CWBIO, Beijing, China).