CD44 specifically interacts with P falciparum invasion ligands EBA-175 and EBA-140. (A) Mass spectrometry results from affinity purification experiments in which bead-immobilized rCD44-Fc or rFc were incubated with lysate from P falciparum strain 3D7 schizont-stage parasites. The heat map shows the normalized spectral counts (based on protein size) of the most abundant P falciparum proteins detected and their enrichment in rCD44-Fc compared with rFc. NaN indicates “no counts.” The table shows log probability, raw number of spectra, and unique peptides, and coverage of EBA-140 and EBA-175 proteins found in the rCD44-Fc lane. (B) Western blot of independent affinity purification experiment in which bead-immobilized rCD44-Fc or rFc were incubated with P falciparum schizont-stage lysate, followed by immunoblotting for EBA-175 or EBA-140 using anti-EBA-175 or anti-EBA-140 rabbit antisera (from Alan Cowman, 1:2000) followed by goat anti-rabbit-HRP secondary antibody (1:25 000). SN, supernatant. (C) Flow cytometry–based binding assays in which recombinant RII of EBA-175-His (left) or EBA-140-His (right) were incubated with beads coated with rCD44-Fc, rFc, or no protein. Binding was detected using a mouse anti–6x-His IgG antibody (Invitrogen; 1:500) and goat anti-mouse IgG-FITC (1:1000). (D) Flow cytometry–based binding assays between rCD55-Fc or rFc and RII of EBA-175-His (left) or EBA-140-His (right). (E) Binding assays of RII EBA-175 (left) or RII EBA-140 (right) with rCD44-Fc after incubation of the protein-coated beads with anti-CD44 mouse monoclonal antibody BRIC 222 (50 μg/mL) or isotype control BRIC 170, which targets the cytoplasmic domain of Band3 (50 μg/mL).