Figure 1.

Association of PfRab5b and PfArf1 GTPases rather than PfRab1b in adjacent to the nucleus. Transformant parasites carrying the PfArf1-RFP (A, red) or PfRab1b-RFP (B, red) constructs with PfRab5b^Q94L-YFP-DD (green) under the dual Pfef1α promoter were stabilized with Shld1, and were then used in the immunofluorescence assay. The fluorescence of RFP and YFP was captured. Arrowheads indicate the colocalization of PfArf1 and PfRab1b with PfRab5b^Q94L. The nuclei were stained with DAPI (blue). Representative images showing mononuclear early trophozoite (upper), two nuclear late trophozoite (middle), and multinucleated early schizont (lower) stages are shown. White arrowheads indicate the colocalization of PfRab5b^Q94L-YFP-DD and PfArf1-RFP or PfRab1b-RFP. The bars indicate 2 µm. (C) Reciprocal immuoprecipitation experiments of PfArf1-RFP via interaction with PfRab5b^Q94L-YFP-DD. PfRab5b^Q94L-YFP-DD and PfArf1-RFP double-expressing parasites were crosslinked with DSP as described in Materials and Methods, and immunoprecipitated with rabbit anti-RFP antibody (IP: +). Immunoprecipitated PfArf-RFP (a white arrowhead) and PfRab5b^Q94L-YFP-DD (a black arrowhead) was visualized with mouse anti-RFP or anti-GFP antibodies, respectively. In the absence of rabbit anti-RFP antibody during immnoprecipitation (IP: −), neither PfArf-RFP nor PfRab5b^Q94L-YFP-DD was detected. Anti-Hsp90 antibody was used as a negative control. Two 50 kDa bands in pull down fraction (an asterisk) were non specific recognition of secondary antibody against anti-rabbit IgG.