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

a, Targeting strategy for creating 3D7-PfGARP KD parasites. b, Immunoblot analysis of 3D7-PfGARP KD. Parasites were sorbitol-synchronized at the ring stage, incubated with or without anhydrotetracycline (ATc) for 20 h and blots were probed with anti-V5. The expected molecular weight of the PfGARP V5-tagged protein is 124 kDa; however, its apparent mobility is 165 kDa owing to its acidic composition. Lane 1, uninfected RBCs; lane 2, RBCs infected with 3D7-PfGARP KD parasites and cultured without anhydrotetracycline; lane 3, RBCs infected with 3D7-PfGARP KD parasites and cultured with anhydrotetracycline. c, d, Expression of PfGARP on the surface of human RBCs infected with 3D7-PfGARP KD parasites. Ring-stage 3D7-PfGARP KD parasites were cultured to the trophozoite stage in the absence (c) or presence (d) of anhydrotetracycline. Expression of PfGARP on the surface of fixed but not permeabilized infected human RBCs was determined by flow cytometry, using monoclonal anti-V5 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 KD parasites. Ring-stage parasites were cultured with or without anhydrotetracycline. Parasitaemia was measured by microscopy. Data are mean ± s.e.m. of three biologically independent replicates. f, GIAs using 10% anti-rPfGARP-A serum or pre-immune serum on 3D7-PfGARP KD parasites cultured with or without anhydrotetracycline. Data are mean ± s.e.m. of three biologically independent replicates. The P value was calculated by non-parametric two-sided Mann–Whitney U-test. Data are representative of three independent experiments (b, d, e, f) or five independent experiments (c).