Figure 2.

PIMMS01 protein expression and localization. (A) Western blot analysis using α-GFP antibody on whole cell lysates of P. berghei c01::gfp parasites. The PbPIMMS01::GFP fusion protein band is indicated with a black arrowhead. The ANKA 2.34 parental parasite line was used as a negative control. Tubulin was used as a loading control. MBS, mixed blood stages; Gc(-), non-activated gametocytes; Gc(+), activated gametocytes; Ook, ookinetes. (B) Western blot analysis using the α-HA antibody on Triton X-100 soluble lysates of in vitro gametocyte (Gc) and ookinetes (Ook) and Triton X-100 insoluble lysates (Gc*, Ook*) of c01::3xha. The c507 reference line (left panel) was used as a negative control. PbPIMMS01::HA fusion protein band is indicated with a black arrowhead. GFP and P28 were used as a loading and a stage specific control, respectively. (C) Immunofluorescence assays on ookinetes of PIMMS01 tagged with GFP (left panel) and 3xHA (right panel) stained with α-GFP or α-HA (green), respectively, as well as with the ookinete surface α-P28 (red). DNA was stained with DAPI. Images are de-convoluted projections of confocal stacks. BF, bright field; Scale bars, 5 μm. (D) Western blot analysis using α-Pfc01 antibody on whole cell lysates of coluzzii midguts at 22 h post blood feeding (hpbf). The PfPIMMS01 protein band is indicated with a black arrowhead. Tubulin was used a loading control for Gc and Sch while Pfs25 was used for 22 hpbf. (E) Immunofluorescence assays of P. falciparum NF54 parasites in mosquito blood bolus at 2 hpbf, ookinetes in mosquito blood bolus at 19 hpbf and ookinetes traversing the mosquito midgut epithelium at 27 hpbf, stained with α-Pfc01 (green) and the female gamete/zygote/ookinete α-Pfs25 (red) antibodies. Pre-immune (pre-imm) serum was used as negative control. DNA was stained with DAPI. Pre-immune serum was used as a negative control. BF, bright field; hpbf, hours post blood feeding. Scale bars, 5 μm.