4. 3 Western blot analysis
For the analysis of the Scd-3XHA fusion protein in transgenic parasites, blood-stage parasites were harvested, washed three times with PBS, and pelleted by centrifugation. The iRBC pellet was lysed using 0.15% saponin, and the released parasites were pelleted, washed with PBS containing complete protease inhibitors (Roche, Switzerland), and resuspended in Laemmli buffer. Immunoblotting was performed as previously described (Narwal et al. , 2022). Briefly, samples were resolved on SDS‒PAGE and transferred onto nitrocellulose membranes (Bio-Rad, USA), blocked with 5% nonfat dry milk in PBS, and incubated with anti-HA antibody (diluted 1:1,000, Novus Biologicals, USA) followed by incubation with HRP-conjugated anti-rabbit IgG (diluted 1:5,000, Amersham Biosciences, United Kingdom). The signals were detected using ECL chemiluminescent substrate (Thermo Scientific, USA) in a ChemiDoc XRS+ System (Bio-Rad, USA).