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. 2011 Nov;79(11):4523–4532. doi: 10.1128/IAI.05412-11

Fig. 5.

Fig. 5.

Erythrocyte binding assay with native and recombinant GAMA. (A) Erythrocyte binding activities of native and recombinant GAMA proteins. The native GAMA protein in the culture supernatant or recombinant proteins (ECTO, Tr1, and Tr3) were incubated with human erythrocytes. The bound proteins were eluted with 0.5 M NaCl in PBS, pH 7.4, either directly from the incubated erythrocytes (E) or from the erythrocytes washed once with iRPMI (W). The eluted protein was detected by Western blotting either with rabbit anti-FL antibody (for GAMA) or with anti-penta-His antibodies (for ECTO, Tr1, and Tr3). For each experiment, the intact protein (without incubation or elution) was also detected by Western blotting as a control (C) (arrowheads). (Given that Tr3 has a cysteine residue, Tr3 forms an artificial homodimer [marked by an asterisk] with erythrocyte binding capacity.) (B) Tr3 competes for binding to a receptor(s) against native GAMA. The bands in lanes 1 through 5 and bands in lanes 6 and 7 refer to Tr3 and native GAMA, respectively, present in the blot of erythrocyte-bound proteins eluted from either controls or different treatments used in the binding assay (described above the lanes; also refer to Materials and Methods). Tr3 and native GAMA in the blot were detected with anti-penta-His and anti-FL antibodies, respectively. (C) GAMA binds erythrocytes in a receptor-specific manner. The erythrocyte binding abilities of native GAMA (present in the culture supernatant) and recombinant Tr3 were tested by incubation with untreated (U), neuraminidase-treated (N), trypsin-treated (T), and chymotrypsin-treated (C) erythrocytes, then elution with 0.5 M NaCl in PBS, pH 7.4, and detection by Western blotting with anti-penta-His (for Tr3) or anti-FL (for GAMA) antibodies. As a control for erythrocyte treatment, native EBA175 in the identical culture supernatant was also examined and detected by anti-EBA175 (regions 3 to 5) antibody. The values indicate percent changes in signal intensity of the relevant band relative to the band in lane U, calculated using Image J.