Figure 4

r Pv RON5 binding to human reticulocytes. (A) Immunoprecipitation and WB assays with reticulocytes (lane 1) and packed RBC (lane 2) with rPvRON5. 15 μg rPvRON5 was incubated with human reticulocytes or packed RBC passed through CF11 columns, followed by separation by dibutyl phthalate gradient. Cell-associated protein was eluted with 1 M NaCl in PBS and the eluted fraction was immunoprecipitated with anti-polyhistidine antibodies. The precipitate was run on an SDS-PAGE gel and revealed with anti-polyhistidine antibodies. (B) Indirect immunofluorescence binding studies. (i) reticulocyte labelling with antibodies against the transferrin receptor (CD71) which is expressed at high levels during erythroid development phases and is absent in mature cells (RBC). (ii) rPvRON5 recognition by polyclonal antibodies directed against the recombinant bound to reticulocytes and RBC. (iii) The cells were seen in white light and those which were positive for CD71 (red). (iv) The cells were observed in white light simultaneously with rPvRON5 binding (green) to cell membrane. (v) Merging between red fluorescence (CD71) and green (rPvRON5). Greater rPvRON5 preference for reticulocytes than mature RBCs was observed.