Visualizing PfDOZI::GFP Localization

The expression of PfDOZI::GFP and its subcellular localization were directly visualized using the GFP signal under the Olympus FV1000 confocal microscope using a 60× oil objective and processed by FV10-ASW imaging software. Nuclei were counter-stained with DAPI. For IFA, parasite cultures were coated on slides, allowed to air-dry, and then fixed by methanol: acetone (1:1) for 20 min at −20 °C. The samples were treated with 0.01% Triton X-100 for 5 min to permeabilize the parasite cell membrane and 5% BSA in PBS for 30 min at room temperature to block non-specific binding sites. Subsequently, the slides were incubated with primary antibodies in 3% BSA/PBS at 4 °C overnight and then secondary antibodies at room temperature for 1 h. Depending on the purpose, the primary antibodies included anti-PABP1 (1:1000)^{32 (link)}, anti-Alba4 (1:1000), anti-α-tubulin II (1:2000) for male gametocytes, anti-GFP (1:500, Invitrogen), anti-β-tubulin (1:250, Sigma), anti-PfAMA1 (1:250, MR4), and anti-PfMSP5 (1:500, MR4). Secondary antibodies were Alexa Fluor 488 or 594 conjugated goat anti-mouse or donkey anti-rabbit IgG antibodies. The slides were mounted with Vectashield antifade mounting medium with DAPI and observed under an Olympus FV1000 confocal microscope. ImageJ software was used to measure the fluorescence density.