Western Blot Analysis of Synchronized Plasmodium Parasites

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.