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

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

Target genes and RT-PCR primers.

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

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

Summary of gene-specific expression patterns.

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

Heterodimerization of mMOR-1 and mSigmar1 splice variants.

<p>Flag-tagged mMOR-1 and HA-tagged mSigmar1 variants were transiently co-transfected into HEK293 cells. Co-IP was performed as described in Materials and Methods. Briefly, cleared whole cell lysate was incubated with EZview Red Anti-Flag or anti-HA Affinity gels overnight. After washing, the tagged proteins on the affinity gels were eluted with 3xFlag or HA peptide, and the elutes were analyzed in Western blot. Top panel: Immunoblot (IB) with anti-HA antibody using elutes from immunoprecipitation (IP) with EZview Red Anti-Flag Affinity Gel. Bottom panel: IB with anti-Flag antibody using elutes from IP with EZview Red Anti-HA Affinity Gel. The representative blots from two independent experiments were shown.</p

Isolation and characterization of alternatively spliced variants of the mouse sigma₁ receptor gene, Sigmar1

<div><p>The sigma₁ receptor acts as a chaperone at the endoplasmic reticulum, associates with multiple proteins in various cellular systems, and involves in a number of diseases, such as addiction, pain, cancer and psychiatric disorders. The sigma₁ receptor is encoded by the single copy SIGMAR1 gene. The current study identifies five alternatively spliced variants of the mouse sigma₁ receptor gene using a polymerase chain reaction cloning approach. All the splice variants are generated by exon skipping or alternative 3’ or 5’ splicing, producing the truncated sigma₁ receptor. Similar alternative splicing has been observed in the human SIGMAR1 gene based on the molecular cloning or genome sequence prediction, suggesting conservation of alternative splicing of SIGMAR1 gene. Using quantitative polymerase chain reactions, we demonstrate differential expression of several splice variants in mouse tissues and brain regions. When expressed in HEK293 cells, all the splice variants fail to bind sigma ligands, implicating that each truncated region in these splice variants is important for ligand binding. However, co-immunoprecipitation (Co-IP) study in HEK293 cells co-transfected with tagged constructs reveals that all the splice variants maintain their ability to physically associate with a mu opioid receptor (mMOR-1), providing useful information to correlate the motifs/sequences necessary for their physical association. Furthermore, a competition Co-IP study showed that all the variants can disrupt in a dose-dependent manner the dimerization of the original sigma₁ receptor with mMOR-1, suggesting a potential dominant negative function and providing significant insights into their function.</p></div