(A) Expression of TfR1, BSG and GYPA on the surface of jkRBCs, TfR1 mutants, ∆BSG null and cultured erythrocytes (cRBCs) as measured by flow cytometry. The right most panels show cytospin analysis of cells stained with May-Grünwald Giemsa staining technique. (B) TfR1∆G217 mutation in TfR1 abrogates PvRBP2b binding as observed using analytical SEC. (C) Quantitative surface proteomics demonstrate specific reduction in TfR1 protein levels in TfR1 mutants compared to wildtype jkRBCs. Levels of Tf, the binding partner for TfR1, are similarly reduced. Significance A was used to estimate p-values, and a minimum of 2 peptides were required for protein quantitation. (D) Binding of recombinant PvRBP2b fragments to jkRBCs, TfR1mutants, ∆BSG and cRBCs cells are shown in blue. Negative controls of unstained cells and isotype control stained cells are shown in the white and orange lines respectively. Compilation of results from PvRBP2b fragment binding to jkRBCs, TfR1 mutants, ∆BSG and cRBCs (right panel). Mean ± S.E.M, n = 3. (E) Comparison of invasion efficiency between jkRBCs and TfR1 mutant cell lines with either P. vivax or P. falciparum. The data shown are averages and the standard error of the mean from between four to five biological replicates shown as open dots. P value was calculated using a paired, two-tailed T test, **** P ≤ 0.0001 and ns is non-significant.