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. 2021 Mar 15;11:634273. doi: 10.3389/fcimb.2021.634273

Figure 3.

Figure 3

PIMMS57 protein expression and localization. (A) Western blot analysis using α-GFP antibody on whole cell lysates of the P. berghei c57::gfp parasites. The PbPIMMS57::GFP fusion protein band is indicated with a black arrowhead. The ANKA 2.34 parental parasite was used as a negative control. Tubulin was used 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 c57::3xha. The c507 reference line (left panel) was used as a negative control. PbPIMMS57::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) Western blot analysis using the α-Pbc57 peptide antibody on whole cell lysates of MBS, Gc, and ookinetes of the c507 parasite line. The PbPIMMS57 protein band is indicated with a black arrowhead. Δc57 parasites were used as a negative control. GFP was used as a loading control. (D) Western blot analysis using the α-Pbc57 antibody on fractionated in vitro ookinetes. The PbPIMMS57 protein band is indicated with a black arrowhead. Δc57 ookinetes were used as a negative control. P28 was used as a loading control. Soluble (Sol), Triton X-100 soluble (TriSol) and Triton X-100 Insoluble (TriInso) fractions are shown. (E) Immunofluorescence assays on ookinetes of PbPIMMS57 tagged with GFP and 3xHA stained with α-GFP or α-HA (green; white arrows) as well as with the ookinete surface α-P28 (red). DNA was stained with DAPI. Staining of the c507 and ANKA 2.34 parental parasites were used as negative controls for the HA and GFP staining, respectively. Images are de-convoluted projections of confocal stacks. BF, bright field; Scale bars, 5 μm. (F) Western blot analysis using α-Pfc57 antibody on whole cell lysates of A. coluzzi midguts at 22 hpbf. The PfPIMMS57 protein band is indicated with a black arrowhead. Pfs25 was used as a loading and stage specific control for 22 hpbf. (G) Immunofluorescence assays of P. falciparum NF54 parasites in mosquito blood bolus at 2 hpbf (top) and ookinetes in mosquito blood bolus at 19 hpbf (bottom), stained with α-Pfc57 (green) and the female gamete/zygote/ookinete α-Pfs25 (red) antibodies. Pre-immune serum was used as a negative control. DNA was stained with DAPI. Staining with pre-immune serum was used as a negative control. BF, bright field; Scale bars, 5 μm.