Figure 4

Overexpression of STEVOR does not promote GIE adhesion to erythroid cells. (a) Western blot analysis of STEVOR expression in stage III GIEs from the transgenic Stevor-Ty1 line cultivated with and without BSD, the transfection control line (Control) and the NF54 clone E10F11. Immunoblots were probed with a rabbit polyclonal antibody directed against STEVOR PF3D7_0300400 protein and with a mAb directed against PfHsp70. Quantification of STEVOR level relative to the loading control PfHsp70 was performed by densitometry (ImageJ software). Decrease of STEVOR expression in Stevor-Ty1 GIE cultivated without BSD indicates a loss of episomal expression of the epitope-tagged STEVOR protein. Full-length blots are presented in Supplementary Fig. S3. (b) IFA of methanol-fixed blood smears showing the expression and erythrocyte membrane localization of PF3D7_1040200 -Ty1 STEVOR protein (red) in Stevor-Ty1 GIE cultivated with and without BSD. GIE were stained with anti-Ty1 mAb followed by anti-mouse Alexa594-conjugated IgG, and parasite nuclei were counterstained with Hoechst 33342. Pictures were taken under identical exposure conditions. Absence of Ty1 expression in Stevor-Ty1 GIE cultivated without BSD indicates a loss of episomal expression of the epitope-tagged STEVOR protein. Bars represent 5 µm. (c,d) Cell-cell adhesion assay of MACS-purified transgenic Stevor-Ty1 GIE cultivated with and without BSD, of transfection control GIE (Control), of a transgenic GIE downregulating STEVOR expression (Null) or of uninfected erythrocytes (uE) to K562 cells (c) or to human primary erythroblasts (d). Error bars denote the standard error of the mean. ns, non-significant differences in adhesion rates. n = number of experiments.