C3b/C4b interaction with CR1 is not perturbed by PfRh4 binding. (A) PfRh4 binding does not affect C3b interaction with sCR1. Duplicate injections of a concentration series of sCR1 (i) or a mixture of sCR1 plus 3-fold molar excess of rPfRh4 (iii) onto a CM5 chip coupled with C3b. K_D measurements of sCR1:C3b complex alone (ii) or in the presence of 3-fold molar excess of rPfRh4 (iv) with fitted value indicated by dotted line (B) PfRh4 binding does not affect C4b interaction with sCR1. Duplicate injections of a concentration series of sCR1 (i) or a mixture of sCR1 plus 3-fold molar excess of rPfRh4 (iii) onto a CM5 chip coupled with C4b. K_D measurements of sCR1:C4b complex alone (ii) or in the presence of 3-fold molar excess of rPfRh4 (iv) with fitted value indicated by dotted line. In all panels, blank-subtracted sensorgrams are shown. (C) Native PfRh4 binding to erythroid CR1 was not inhibited by C3b or C4b. Competitive binding assays were performed by incubating either C3b or C4b with invasion supernatants at the stated final concentrations (8 μg/mL or 16 μg/mL). Immunodetection of parasite proteins with anti-PfRh4 and anti–EBA-175 antibodies after erythrocyte binding is shown. (D) PfRh4 invasion pathway was not inhibited in the presence of C3b or C4b. Parasite strains W2mefΔRh4 (gray bars) and 3D7 (black bars for untreated, white bars for nm-treated) were tested in growth assays in the presence of 0.5 mg/mL C3b or C4b. Growth (percentage of control) on the y-axis refers to the percentage parasitemia in the presence of CR1 constructs relative to the percent parasitemia with the addition of PBS (arbitrarily set to be 100%).