Figure 6: Loss of TGIF affects RON2 synthesis and trafficking.

(A) Left. Schematic of the pulse-chase recycling assay. Right. Immunofluorescence showing recycled RON2 stained with Halo-TMR (green) in TGIF-Ty:RON2-Halo parasites grown for 24 hours with ethanol (control) or IAA (knockdown). Scale bars are 5μm. Representative images from two experimental replicates (N = 16 vacuoles per condition). (B) Schematic of the pulse-chase trafficking assay. (C) TGIF-Ty:RON2-Halo parasites labeled with HaloLigand-TMR (green) and HaloLigand-JF646 (magenta) after growth in ethanol (control) or IAA (knockdown). Time points indicate period of growth between 1^st and 2^nd labeling reactions. Scale bars are 5μm. Representative images from three experimental replicates (N > 300 vacuoles per time point per condition). (D) Stacked bar chart indicating the localization of newly synthesized RON2 at each time point from (C). P-value indicated. (E) Bar graph indicating the percent of vacuoles with JF646 staining at each time point, indicating the presence of newly synthesized protein. P-value indicated. (G) Illustration of RON2 trafficking pathway in control and TGIF knockdown parasites.