Protein patterns of <i>S. mansoni</i> organs/tissues and adults.

<p>1.2 µg total protein from male (Mars symbol), female (Venus symbol), testes (Te), ovaries (Ov), and male tegument (T) were separated by 13% SDS-PAGE and visualised by silver staining. Marker (M) = PageRuler Plus Prestained Protein Ladder (Fermentas).</p

Summary of gene-specific expression patterns.

<p>* = current study, <b>+</b> = detected, <b>−</b> = not detected, <b>nd</b> = not determined.</p

Target genes and RT-PCR primers.

<p>Target genes and RT-PCR primers.</p

Quantitative and qualitative microfluid analysis of total RNA.

<p>RNA-analyses exemplarily shown for RNA isolated from adult males (<b>A</b>), testes (<b>B</b>), and ovaries (<b>C</b>) obtained by the organ isolation procedure were used. The figure shows a “gel-like image” consisting of the RNA-ladder and the appropriate total RNA sample (left) and the corresponding electropherogram (right); fluorescent units (FU), retention time (s).</p

Gonad protein-specific immunoblots.

<p>15 µg of total protein per lane isolated from adult worms (Mars and Venus symbol), testes (Te), ovaries (Ov), and tegumental proteins of both genders (T) were analysed by immunoblotting employing immune sera directed against SmSPRM1hc (Permease 1 heavy chain), SmHSP70 (Heat shock protein 70), SmAQP (Aquaporin), and SmFKBP12 (FK506-binding protein); for references see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd-0002336-t001" target="_blank">Table 1</a>.</p

Schematic illustration and bright-field microscopy (BF) of gonad tissues following tegument solubilisation and protease treatment.

<p><b>A)</b> Crude preparation of intact testes (TE) together with a part of an incompletely digested male worm body (MB) and different types of cells (CE) (left) and an mature ovary (Om) surrounded mainly by S4-vitelline cells (VC) from the vitellarium (right); immature ovary (Oi) and ootype (OT) with vitelloduct (VD) and oviduct (OD) isolated from a unisexual female; the ootype was contrasted by brief staining with Ponceau S; asterisk: “hymen”-like morphological structure typical for ootypes of unisexual females <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd.0002336-Beckmann1" target="_blank">[18]</a>, <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd.0002336-Beckmann2" target="_blank">[108] </a><b>B)</b> Mechanical transfer by pipetting led to the enrichment of pure testes (TE), mature ovaries (Om) after collecting and concentrating. TL (testes lobe), Op (ovary - posterior part containing mature primary oocytes in the case of mature ovaries), Oa (ovary - anterior part containing immature, stem cell-like oogonia); vitellarium (VI) with vitelline lobes (VL); dashed arrow = continued from <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd-0002336-g001" target="_blank">Figure 1</a>.</p

Schematic illustration and surface electron microscopy (SEM)-analyses of tegument solubilisation by TS-solution treatment.

<p><b>A)</b> Untreated control males (upper left) and females (upper right) showing intact tegument (TE) with spines (SP), pits (PI), and sensory endings (SE). <b>B)</b> The tegument was completely removed due to detergent treatment exposing the outer circular muscles (CM) and the basis of the (male-specific) tubercles (TU). Membranocalyx (MC), plasma membrane (PM), longitudinal muscles (LM), basal membrane (BM), musculature (MU), parenchyma (PA); modified according to <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd.0002336-Braschi3" target="_blank">[107]</a>; dashed arrow = continued in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd-0002336-g002" target="_blank">Figure 2</a>.</p

SmShb interacts with ligand-activated SmVKR1 and SmVKR2 receptors.

<p>(A) cRNA encoding V5-tagged SmVKR1 WT or SmVKR1 Y₉₇₉F were injected with or without cRNA encoding HA-tagged SmShb in <i>Xenopus</i> oocytes. Following their expression, full-length receptors were activated by L-Arg to induce their auto-phosphorylation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163283#pone.0163283.ref011" target="_blank">11</a>]. Proteins were then analysed by immunoprecipitation (IP) using anti-V5 or anti-HA antibodies followed by western blot (WB) using anti-V5, anti-HA or PY20 (anti-tyrosine phosphorylation) antibodies. Results demonstrated that only phosphorylated SmVKR1 WT bound to and phosphorylated SmShb as revealed by PY20 antibodies. (B) cRNA encoding V5-tagged SmVKR2 WT or SmVKR2 F₉₄₉Y were injected with or without cRNA encoding HA-tagged SmShb in <i>Xenopus</i> oocytes. Following their expression, full-length receptors were activated by Ca⁺⁺ to induce their auto-phosphorylation [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163283#pone.0163283.ref011" target="_blank">11</a>]. Proteins were then analysed by immunoprecipiration (IP) using anti-V5 or anti-HA antibodies followed by western blot (WB) analysis using anti-V5, anti-HA or PY20 (anti-tyrosine phosphorylation) antibodies. Results indicated that phosphorylated SmVKR2 WT did not bind SmShb whereas the mutated V5-SmVKR2 F₉₄₉Y bound to and phosphorylated SmShb.</p

Gonad-RNA specific RT-PCRs.

<p>Total RNA of testes (Te), ovaries (Ov) and adult couples (merged Mars/Venus symbol) was isolated by Trizol and reverse transcribed. RT-PCRs were performed using gene-specific primers targeting SmHSP70 (Heat shock protein 70), SmFKBP12 (FK506-binding protein), SmCNA (Calcineurin subunit A), SmTGFβRI (Transforming growth factor β receptor I), SmAxDynIC (Axonemal dynein intermediate chain), SmAQP (Aquaporin), SmSPRM1hc (Permease 1 heavy chain), and SmNPP-5 (Nucleotide pyrophosphatase/phosphosdiesterase type 5); for references see <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd-0002336-t001" target="_blank">Table 1</a>. Marker (M) = Hyperladder I (Bioline). Target genes depicted in green were also analysed by immunoblotting (<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002336#pntd-0002336-g007" target="_blank">Figure 7</a>).</p

Co-Immunoprecipitation confirms complex formation.

<p>Co-Immunoprecipitation of HA-tagged Smβ-Int1 and V5-tagged SmILK (about 56 kDa), SmPINCH (about 42 kDa), and SmVKR1 (about 150 kDa) expressed in <i>Xenopus</i> oocytes. Anti-HA antibodies immunoprecipitated Smβ-Int1 together with SmILK, SmPINCH, and SmVKR1 upon co-expression in oocytes only when their wildtype forms were used (lane 11). Using deletion variants (DEL) of SmILK or SmPINCH, no immunoprecipitated interaction partners were detected (lanes 12, 13). No immunoprecipitated molecules were detected in further controls including individual molecules (lanes 1–6) or combinations with three of the four potential binding partners (lanes 7–10).</p