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).