561979.fig.006
Figure 6: TvLEGU-1 binds to the surface of fixed and live HeLa cells. (A) Coomassie brilliant blue-stained protease-rich extracts from parasites grown in iron-rich conditions (lane 1). WB assay of eluted proteinases after cell-binding assay of protease-rich extracts with fixed HeLa cells (lane 2) or mock HeLa cells (lane 3) incubated with the anti-TvLEGU-1r antibody (1 : 1 000 dilution). (B) The TvLEGU-1r protein interacts with fixed HeLa cells. Coomassie brilliant blue-stained TvLEGU-1r protein (lane 1) and TvLEGU-1r protein eluted after cell-binding assays with fixed HeLa cells (lane 2). Coomassie brilliant blue-stained bovine serum albumin (BSA) (lane 3) and BSA eluted after cell-binding assays with fixed HeLa cells (lane 4) used as a specificity control; mock experiment with fixed HeLa cells (lane 5). (C) Recognition of TvLEGU-1r by the anti-CP30 antibody. Coomassie brilliant blue-stained TvLEGU-1r (lane 1); WB assay of the TvLEGU-1r protein incubated with the anti-CP30 (1 : 5 000 dilution) [7] or the anti-TvLEGU-1r antibody (1 : 10000 dilution), or PI serum (1 : 1 0000 dilution) used as a negative control (lanes 2, 3, and 4, resp.); kDa, molecular weight markers in kilodaltons. Protein bands were visualized by SDS-PAGE on 10% polyacrylamide gels. Arrowheads show the position of TvLEGU-1 (A), BSA (B), or TvLEGU-1r ((B) and (C)) proteins. (D) Confocal microscopy images after immunofluorescence assays using the anti-TvLEGU-1r antibody with fixed HeLa cells incubated with the TvLEGU-1r protein (a, b, and c). Fixed HeLa cells directly incubated with the anti-TvLEGU-1r antibody were used as negative controls. Conjugated anti-rabbit IgG-FITC was used as a secondary antibody (1 : 100 dilution) (b and e). Nuclei stained with DAPI (a and d). Merge and bars: 20 μm (c and f). (E) Confocal microscopy images after immunofluorescence assays using the anti-TvLEGU-1r antibody with live HeLa cells incubated with the TvLEGU-1r protein (a, b, and c). Live HeLa cells were directly incubated with the anti-TvLEGU-1r antibody and used as negative controls (d, e, and f). Conjugated anti-rabbit IgG-FITC was used as a secondary antibody (1 : 100 dilution) (b and e). Parasite membranes were labeled with DIL (a and d). Nuclei labeled with DAPI, merge, and bars: 18 μm (c and f).