Extended Data Fig. 4: Construction and characterization of the 3D7-PfGARP KO parasite line.

a, Targeting strategy for creating 3D7-PfGARP KO parasites, b, Immunoblot analysis of 3D7-PfGARP KO. Trophozoite-stage 3D7 wild-type or 3D7-PfGARP KO parasites were probed with anti-PfGARP, and with anti-histone H3 as a loading control. Lane 1, RBCs infected with 3D7-PfGARP KO parasites; lane 2, RBCs infected with 3D7 wild-type parasites. c, d, Expression of PfGARP on the surface of human RBCs (fixed but not permeabilized) infected with 3D7-PfGARP KO parasites. Ring-stage stage 3D7 wild-type or 3D7-PfGARP KO parasites were cultured to the trophozoite stage. Expression of PfGARP was determined by flow cytometry, using anti-PfGARP as the primary and anti-mouse IgG–Alexa Fluor 488 as the secondary antibody. Infected RBCs were gated and identified as described in Fig. 1e. e, Growth curves for 3D7-PfGARP KO parasites. Ring-stage 3D7 wild-type or 3D7-PfGARP KO parasites were plated at 0.5% parasitaemia and cultured for 6 days. Parasitaemia was measured by microscopy. Data are mean ± s.e.m. of three biologically independent replicates. f, Ring-stage 3D7 wild-type or 3D7-PfGARP KO parasites with targeted deletion of PfGARP were cultured at a 1:10 dilution in the presence of anti-PfGARP-A mouse serum that was generated by immunizing mice with PfGARP-A-mRNA LNPs. Negative controls were no antiserum (blue) and pre-immune mouse serum (red). Parasites were cultured for 48 h at 37 °C and ring-stage and early-trophozoite-stage parasites were counted by microscopy. Data are mean ± s.e.m. of six biologically independent replicates. P values were calculated by non-parametric two-tailed Mann–Whitney U-test. Data are representative of two independent experiments.