Figure 4

PfA-M1 [1-30]-GFP yields a 32-kDa protein strictly associated with the parasite membranes that localizes to the ER. A. Soluble proteins (lanes 2 and 3) and membrane proteins (lanes 4 and 5) from parasites expressing PfA-M1-GFP chimera (51, lanes 2 and 4) or PfA-M1 [1-30]-GFP chimera (lanes 3 and 5) were separated by SDS-PAGE, transferred to nitrocellulose and immunoblotted with anti-GFP antibodies as described in the Methods section. PfA-M1-GFP chimera was detected at ~150-kDa in soluble (lane 2) and membrane (lane 4) fractions of the parasites while the ~32-kDa PfA-M1 [1-30]-GFP fusion protein is strictly insoluble (lane 5). Note that free soluble GFP is produced from PfA-M1-GFP chimera (lane 2), but not from PfA-M1 [1-30]-GFP chimera (lane 3). Mw markers in kDa are in lane 1. B. Live imaging of FcB1 parasites transfected with a construct allowing expression of PfA-M1 [1-30]-GFP chimera. Top row: ring stages; bottom row: young schizont stages. Nuclei were stained with Hoechst 33342, 4 μg.ml^-1. cc stands for "colour combine". Scale bar, 5 μm. C. Immuno-fluorescence analysis of FcB1 parasites transfected with a construct allowing expression of PfA-M1 [1-30]-GFP chimera, fixed by using 3.7% formaldehyde and analysed as previously described [4, 6] by using rabbit anti-PfBip [21](red) and mouse anti-GFP (green) antibodies. Top row: trophozoite stages; bottom row: schizont stages. Nuclei were stained with Hoechst 33342, 4 μg.ml^-1. cc stands for "colour combine". Scale bar, 5 μm.