Expression of PfCLK-3 and PfCLK-4 in the cytoplasmic and nuclear fractions of blood stage parasites.

<p>Western-blot analyses on lysates of mixed blood stage parasites (BSP) as well as of nuclear pellet (NP) or cytoplasmic fraction (CF) of BSP using rat antisera against PfCLK-3 or mouse antisera against PfCLK-4 detected the full length protein bands for PfCLK-3 and PfCLK-4 of approximately 80 and 150 kDa, respectively. For PfCLK-4, two additional protein bands of approximately 100 and 70 kDa are present. No proteins were detected by the anti-PfCLK antisera in lysates of non-infected erythrocytes (EC). Immunoblotting with sera from non-immunized rat (NRS) or mouse (NMS) did not result in the labelling of any protein bands.</p

Antiplasmodial and transmission blocking activities of the CLK inhibitors.

<p>Antiplasmodial and transmission blocking activities of the CLK inhibitors.</p

Localization of the SR proteins in the parasite nucleus.

<p>Transforming trophozoites (2-nuclei stages) were prepared for IFA as described in Fig. 5 and the localization of the SR proteins was investigated in the Hoechst-positive parasite nuclei. Bar, 5 µm.</p

Effect of CLK inhibitors on blood stage parasites.

<p>A. Stage of growth inhibition of asexual blood stages between 12–60 h of CHX treatment. CHX at approximate IC₅₀ and IC₈₀ concentrations or 0.5% vol. of DMSO was added to synchronized parasites at the ring stage. Giemsa stained blood smears were prepared at six time points between 0–60 h of incubation with CHX and the numbers of ring stages, trophozoites, schizonts and dead parasites were counted. Histograms indicate the percentages of developmental stages present in the respective blood smears. B. Parasites were treated with CLK inhibitors as described above and the stage of growth inhibition was determined at 24 h of compound incubation. A total number of 100 parasites were counted in A and B for each condition. C. Compounds at IC₅₀ concentrations or 0.5% vol. of DMSO were added to stage II gametocyte cultures for two days. After seven days, the numbers of stage IV and V gametocytes were counted in a total of 1000 erythrocytes and correlated to the gametocyte numbers of DMSO control (set to 100%). *, significant reduction of gametocyte numbers (p&lt;0.001, student’s t-test). Epoxomicin was used as positive and chloroquine was used as a negative control.</p

Phosphorylation of plasmodial SR proteins by immunoprecipitated PfCLKs.

<p>A. Kinase activity assays were deployed to detect phosphorylation of recombinant PfSRSF12 and PfSFRS4 (∼73 and 65 kDa, respectively; indicated by arrows) by two or more of the PfCLKs (autoradiogram; upper panel). B. The N-terminal part (∼95 kDa; indicated by arrows), but not the C-terminal part (86 kDa) of recombinant PfSF-1 was phosphorylated by immunoprecipitated PfCLKs. An additional phosphorylation signal of truncated N-terminal PfSF-1 was visible at approximately 60 kDa. C. MaBP-tag alone (43 kDa) as substrate was used as negative control. Shown here is an assay using PfCLK-3-specific immunoprecipitate, similar results were obtained with immunoprecipitates of other PfCLKs (not shown). Coomassie blue staining (lower panels) of radiolabelled SDS gels was used as a loading control.</p

Subcellular localization of PfCLK-3 and PfCLK-4 in the blood and gametocyte stages.

<p>Mixed asexual blood stage cultures containing trophozoites (TZ) and schizonts (SZ) and mature gametocyte (GC) cultures were fixed with methanol and prepared for IFA, using rat antisera against PfCLK-3 and mouse antisera against PfCLK-4 (green). The parasite nuclei were highlighted by Hoechst staining (blue). The asexual blood stages were labelled with rabbit antisera against PfMSP-1 and gametocytes with rabbit antisera against Pfs230 (red). Bar, 5 µm.</p