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Fig. 3 | Malaria Journal

Fig. 3

From: Identification of a PH domain-containing protein which is localized to crystalloid bodies of Plasmodium ookinetes

Fig. 3

Construction of CryPH-GFP expressing or CryPH gene depleted transgenic parasites. a Western blot analysis for confirmation of gene modification in isolated parasites. Wild-type (WT) or genetically modified (CryPH-GFP or ∆CryPH) PyXNL parasite lines cultured in vitro were collected at indicated time points. These parasites were analyzed by Western blot using anti-CryPH antibodies and anti-Pys25 antibodies under non-reducing conditions. Left panels, a clear protein signal of the size comparable to that of native PyCryPH (indicated by a black arrow) was detected in WT ookinete lysate. Middle panels, one major band of about 60 kDa corresponding to the predicted size of CryPH-GFP (indicated by a green arrow) was revealed in CryPH-GFP expressing transgenic ookinetes. Note that there was no signal of the size of native PyCryPH. Right panels, disappearance of PyCryPH signal in ookinetes with genetically disrupted PyCryPH showed that CryPH expression was completely depleted. In contrast, Pys25 signal intensity was increased during culturing, suggesting that ∆CryPH ookinetes formed normally (lower panel, indicated by an open arrow head). b Detection of CryPH-GFP by immunofluorescence analysis. Cultured CryPH-GFP parasites were stained using anti-GFP antibodies (green) and anti-Pys25 antibodies (red). Nuclei were stained by DAPI (blue). Representative immunofluorescence assay images for parasites at each stage (upper panels) and corresponding bright field images (lower panels) are shown. Bars: 5 µm. c The number of CryPH positive bodies was counted in a total of 300 CryPH-GFP expressing parasites. As the development of sexual stage parasites progressed from zygote, retort, to mature ookinete, the number of cell bodies expressing CryPH increased. Color codes in the column indicates the number of round bodies per parasite

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