Sab4800032

Sample preparation for western blot analysis was performed as previously described (Liu et al., 2020 (link)). Briefly, schizonts were released from erythrocytes with 0.15% saponin, resuspended with an equal volume of 2 × SDS–polyacrylamide gel electrophoresis (PAGE) protein loading buffer, and then heated for 5 min at 100°C before storing at -80°C. Proteins were separated by 10% SDS-PAGE, and then transferred to Immobilon-P transfer membranes (Millipore). Subsequent antibody incubation and membrane wash followed standard procedures. The primary antibodies used in this study included mouse anti-ty1 (Sigma, SAB4800032) at 1:1,000 and rabbit anti-aldolase (Abcam, ab207494) at 1:2,000. The horseradish peroxidase (HRP) conjugated secondary antibodies were used at 1:5,000, including goat anti-mouse IgG (Abcam, ab97040) and goat anti-rabbit IgG (Abcam, ab205718). HRP signals were detected using the ECL western blotting kit (GE healthcare). Especially, pfap2-exp2-ty1-glms parasites were tightly synchronized to a 5-h window and ring-stage parasites were diluted at 0.5% parasitemia with 2% hematocrit in the presence or absence of 5 mM glucosamine. At the second generation, 200 μL of schizont-staged samples were collected for western blotting.

The immunofluorescence assay was performed to detect the localization of PfAP2-EXP2 as described previously (Jing et al., 2018 (link)). Parasites were harvested, released, fixed by 4% paraformaldehyde (Electron Microscopy Sciences) at room temperature for 10 min, and washed by PBS. Prepared samples were incubated with the primary antibodies against ty1 (Sigma, SAB4800032) or GFP (Abcam, ab290) at 1:500 to 1:1,000, followed by the secondary antibodies AlexaFluor 488 goat anti-mouse IgG (ThermoFisher Scientific, A11029) or AlexaFluor 568 goat anti-rabbit IgG (ThermoFisher Scientific, A11036) at 1:500. Preparations were visualized with a Nikon A1R microscope at 60-100 × magnification and images were acquired with NIS Elements software and processed using Adobe Photoshop.