Fig. 2
PyCryPH is expressed in zygotes and ookinetes. a Expression of recombinant CryPH using the wheat germ cell-free protein expression system. Predicted size of the recombinant CryPH is indicated by an arrowhead. b Expression profile of CryPH examined by Western blotting analysis using extracts from in vitro ookinete culture. The same volumes of cultured parasites were taken at the times indicated above. Affinity purified anti-CryPH antibodies were used both under non-reducing (left panel) or reducing (right panel) conditions. The representative stages of parasites at each time point were as follows: 0 h, gametocytes and gametes; 1 h, zygotes; 4 h, retorts; and overnight incubation (O/N), mature ookinetes. The arrow indicates the bands corresponding to CryPH. The same filter was probed with anti-Pys25 antibodies, a zygote/ookinete marker (Lower Panel). Open arrowhead indicates Pys25. c Expression comparison of CryPH in all infective stages. Protein lysates of schizonts, ookinetes, and oocyst-derived sporozoites (1 × 105) were separated by SDS-PAGE and CryPH expression, indicated by an arrow, was detected by Western blotting using anti-PyCryPH antibodies. Protein loading was assessed by re-probing with anti-RAMA antiserum (lower panel, arrow head). d Localization analysis of CryPH using immunofluorescence assay. CryPH was detected as clear circular spots in the parasite cytosol, and the signal intensity increased during ookinete development. Zygotes and ookinetes (retorts and mature ookinetes) cultured in vitro were fixed with acetone for immunofluorescence assays. CryPH signals were associated with malaria pigments. Merge, merged image of CryPH (green), Pys25 (red), and nuclei stained by DAPI (blue). Bars, 5 µm