Parasite morphology and global protein phosphorylation pattern of PKG inhibitor-treated <i>P. falciparum</i> schizonts.

<p>(<b>A</b>) Immunofluorescent staining of DMSO/compound 1-treated WT schizonts using antibodies detecting (i) PfGAP45 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoman1" target="_blank">[34]</a>, (ii) PfSUB1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoh1" target="_blank">[14]</a> and (iii) PfAMA1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Harris1" target="_blank">[23]</a>. Representative images are shown for each staining together with parasite nuclei stained with DAPI. Bars ∼5 µM. (<b>B</b>) Metabolic labelling of phosphoproteins in <i>P. falciparum</i> schizonts. Autoradiographs of (i) 3D7 WT and (ii) gatekeeper mutant 3D7 PfPKG_T618Q schizonts, treated with ³²P-orthophosphate and DMSO (−) or compound 2 (+) prior to lysis, ÄKTA anion exchange chromatography (fractions 10–14 are shown) and separation by SDS-PAGE. Rectangular boxes highlight bands that show a differential signal following inhibitor-treatment in WT, but not PfPKG_T618Q schizonts.</p

PfPKG expression peaks in late blood stages and is carbonate-soluble.

<p>(<b>A</b>) Western blots of synchronised cultures of the PfPKG-HA-3A clone and WT parasites (3D7 clone), 24 hours (mostly mid trophozoites), 30 hours (mostly late trophozoites), 41 hours (mostly early schizonts) and 46 hours (mostly late schizonts) post invasion were detected with anti-HA and anti-humanPKG, respectively. Blots were re-probed with an antibody against Pfαtubulin to estimate the relative total protein loading between lanes. (<b>B</b>) Sequential solubilisation of parasite proteins from saponin-released late trophozoites and schizonts. S1: soluble protein fraction (5 mM Tris-HCl, freeze thaw); S2: peripheral membrane fraction (extraction with 100 mM Na₂CO₃); S3: integral membrane fraction (extraction with 4% SDS/0.5% TX-114/0.5×PBS). Equal volumes of the three supernatants were analysed by SDS-PAGE and Western blots were probed for the integral membrane protein PfPMV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Klemba1" target="_blank">[31]</a>, stripped and re-probed simultaneously for PfGAPDH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Daubenberger1" target="_blank">[30]</a> and PfPKG-HA. Densitometric analysis of the scan of the blot presented revealed that 89.3% of PfPKG-HA is present in fraction S1, while fractions S2 and S3 contain 9.5% and 1.2%, respectively. (<b>C</b>) Immunofluorescent anti-HA detection in fixed smears of erythrocytic stages of the PfPKG-HA-3A clone. Representative images of (i) a ring stage parasite, (ii) three early trophozoites, (iii) an early schizont, (iv, v) late schizonts (approximate hours post invasion: (i) 4–10, (ii) 20–26, (iii) 33–39, (iv, v) 45–48) and (vi) a stage III gametocyte are shown together with bright field images (first column) and parasite nuclei stained with DAPI (second column). Bars ∼5 µM.</p

Subcellular location of PfPKG in mature schizonts.

<p>Dual immunofluorescent detection of PfPKG-HA in fixed smears of early and late schizonts of the PfPKG-HA-3A clone together with (<b>A</b>) PfGAPDH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Daubenberger1" target="_blank">[30]</a>, (<b>B</b>) PfBiP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-vanDooren1" target="_blank">[32]</a>, (<b>C</b>) PfPMV <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Klemba1" target="_blank">[31]</a>, (<b>D</b>) PfRab11A <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-AgopNersesian1" target="_blank">[33]</a> and (<b>E</b>) PfGAP45 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Yeoman1" target="_blank">[34]</a>. Representative images are shown for each antibody, together with bright field images (first column) and parasite nuclei stained with DAPI (in the merged image). Bars ∼5 µM. To quantify co-localisation, Pearson coefficients <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048206#pone.0048206-Manders1" target="_blank">[36]</a> of the individual stains were calculated using Imaris image analysis software (Bitplane).</p