Semi-Quantitative RT-PCR Analysis of SmGCP mRNA

<p>The bottom panel shows the agarose gel separation of the PCR products of SmGCP (bottom), and the constitutively transcribed control, α-tubulin (top). Panel A: Lanes are numbered and the respective stages are listed at the bottom of the panel. Panel B: Adult worm pairs (42-d-old) were left untreated (lane 1) or treated with human TGF-β1 (1 nM; lane 2) or human BMP2 (5 nM; lane 3). Top of each panel shows a bar graph representation of the relative PCR band intensities (%) of SmGCP compared to that of α-tubulin control. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).</p

The Transduction of TGF-β Signal to SmSmad2 via Activated SmTβRII/SmTβRI Receptor Complex, In Vitro: Co-immunoprecipitation of SmSmad2-MH2 and SmSmad4

<p>³⁵S-labeled, in vitro translated products of SmSmad2-MH2 (panel A) and SmSmad2-MH2/AAA (panel B) were incubated with SmSmad4 in the presence of SmTβR-I (wt) and SmTβR-II in the presence or absence of TGF-β1 (1.0 nM) or BMP2 (5.0 nM). Radiolabeled, in vitro translated products were co-precipitated with SmSmad4, using anti-SmSmad4-linker IgG and Protein A Sepharose beads (Amersham Biosciences). Background precipitation was removed by treating ³⁵S-labeled in vitro translated products with anti-SmSmad4-linker IgG and Protein A Sepharose beads. The pre-cleared lysates were then used in the above-described reactions. A positive control reaction (lane 3) was included, in which SmSmad2-MH2, or the AAA mutant peptide, were reacted with SmSmad4 in the presence of the active mutant form of type I receptor, SmTβR-I (Q-D). Reactions, which contain either SmSmad2-MH2 or its AAA mutant form with SmSmad4 in the presence of wild-type SmTβRI, represent the negative controls of the assay (lane 4). Immunoprecipitated products were separated by SDS-PAGE and subjected to autofluorography. Lanes are labeled to specify the input components of each reaction. In vitro translated products (20% of input) are shown (lane 1). Percentage values of precipitated reactive radiolabeled product of each reaction are shown at the bottom of each lane.</p

Whole-Mount S. mansoni Adult Worm Immunofluorescence

<p>S. mansoni adult worms were probed with anti-SmTβRII rabbit IgG and pre-immune rabbit IgG (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020054#ppat-0020054-sg002" target="_blank">Figure S2</a>), followed by biotin-conjugated anti-rabbit IgG. Reactive complexes were detected using streptavidin Alexa Fluor 647 conjugate and analyzed with a Bio-Rad MRC1024 confocal laser microscope. Anti-SmTβRII reactivity is shown in a live male worm (♂) (panels A–C) in different laser sections in tubercles (T) (panel B) and gynaecophoric canal (G) (panel C). Specific surface fluorescence is also shown in live female worms (♀) (panels F and I), whereas green fluorescence fields show the non-specific auto-fluorescence in vitellaria (V), oviduct (OvD) (panel E), and ova (Ov) (panels E and H). An acetone-fixed male worm (♂) shows anti-SmTβRII reactivity in the gynaecophoric canal, oral (Os) and ventral suckers (Vs) (panel K), and in esophagus (O) (panel L). Panels A, D, G, and J are phase-contrast fields of the fluorescent fields B and C, E and F, H and I, and K and L, respectively.</p

In Vitro Interaction of Amino- (N-) Terminal Domains of SmTβRI and SmTβRII

<p>Interaction of the in vitro translated non-labeled N- terminal domain of SmTβRII with ³⁵S-labeled N-terminal domain of SmTβRI, in the presence or absence of different TGF-β ligands (panels A and B) or varied amounts of different TGF-β₁ (panel B). Ligand concentrations (in nM) are shown at the top of each lane. Reactions were precipitated using S-protein agarose beads (EMD Biosciences, Novagen). Precipitated products were separated by SDS-PAGE and subjected to autofluorography. In vitro translated products (5% and 20 %) of input radiolabeled products are shown in the left lane of panels A and B, respectively. Background precipitation of ³⁵S-labeled-SmTβRI-N by S-protein agarose beads is shown in lane 2, panel A. The background reactivity was removed in later experiments by pre-clearing ³⁵S-SmTβRI-N by treatment with the S-protein agarose beads (panel B) prior to use in interaction assay. Percentage values of precipitated reactive radiolabeled product of each reaction are shown at the bottom of each lane.</p

Localization of SmTβRII mRNA Transcripts in Tissue Sections of S. mansoni Adult Worms by FISH

<p>Column A represent phase-contrast fields, Column B represent non-specific autofluorescence fields observed in vitelline lobules (V) using green fluorescence filter (522 nm) and Column C shows specific probe reactivity as represented by far-red fluorescence using 680 nm filter. Row (I) shows sections of a male (♂) and a female (♀) worm probed with the positive control cRNA probe (the antisense strand of eggshell protein P14). Specific fluorescence could be observed in the vitellaria (V) of the female worm. As expected, no specific fluorescence could be observed in male worm sections. Specific reactivity of SmTβRII antisense probe could be seen in vitelline cells (V) and gut epithelial cells (G) in a female worm section (panel IIC) and in subtegumental cells (STC) in a male worm section (panel IIIC). No significant fluorescence could be seen in the negative control reaction using SmTβRII sense cRNA strand (unpublished data).</p

Genomic Structure of S. mansoni TGF-β Type II Receptor Gene

<p>A schematic representation of the genomic locus of S. mansoni TGF-β receptor II (middle) and the two alternatively spliced transcripts, <i>SmTβRII</i> (top) and <i>SmRK2</i> (bottom). The sizes of introns are represented in kb and that of exons (represented as cylinders) are in bp. The red exon is unique for <i>SmRK2</i> and the blue exon is unique for <i>SmTβRII</i>. Block arrows represent the two receptor isoforms, <i>SmRK2</i> (red) and <i>SmTβRII</i> (blue) with locations of start and stop codons indicated.</p

Silencing of TGF-β–Induced Expression of SmGCP by Knocking Down SmTβRII Expression

<p>Semi-quantitative RT-PCR analyses for transcripts of SmGCP as well as various components of schistosomal TGF-β signaling pathways in 35-d-old and 28-d-old old worm pairs, untransformed and transformed with SmTβRII-siRNA, and either left untreated or treated with TGF-β1 (1 nM). The top panel shows the agarose gel separation of the PCR products of SmTβRII (panel B), SmTβRI (panel C), SmGCP (panel D), SmSmad4 (panel E), SmSmad2 (panel F), SmSmad1 (panel G), and the constitutively transcribed control, α-tubulin (panel A). The lanes are labeled to show detailed treatment of each sample. The bar graph representation shows the percentage values of the optical densities in pixels of the PCR bands for each gene compared to the corresponding band of α-tubulin control from the same stage. Values were calculated from three independent PCR amplifications (Error bars represent the standard deviation).</p

Semi-Quantitative RT-PCR Analysis of SmTβRII

<p>Top panel shows agarose gel separation of the PCR products of SmTβRII (middle), SmTβRI (bottom) and the constitutively transcribed control, α-tubulin (top). Lanes are numbered and the respective stages are listed at the bottom of the bar graph. The bar graph representation shows the percentage values of the optical densities in pixels of the PCR bands of SmTβRII (gray bars) and SmTβRI (white bars) compared to the corresponding band of α-tubulin control from the same stage. Values were calculated from three independent PCR amplifications (Error bars represent standard deviation values). Samples included in the assay were the hepato-pancreas regions of the intermediate host; uninfected and 30-d–infected B. glabrata snails, which represent different stages of daughter sporocysts; parasite eggs obtained from the liver of Syrian golden hamsters infected with 5,000 cercariae; cercariae shed and collected from infected snails; and stages representing different time points during schistosome development in mammalian host, obtained by perfusion of infected hamsters for the specified time (15–45-d-old worms). Adult male and female worms, separated after perfusion, were also included in the assay. Samples were subjected to total RNA extraction and cDNA synthesis, followed by PCR amplification using specific primer pairs.</p

Analysis of genes shared with Deuterostomia and with possible roles in host interactions-3

<p><b>Copyright information:</b></p><p>Taken from "Analysis of genes shared with Deuterostomia and with possible roles in host interactions"</p><p>http://www.biomedcentral.com/1471-2164/8/407</p><p>BMC Genomics 2007;8():407-407.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2194728.</p><p></p>Maximum Likelihood tree constructed from the alignment of SmVASLv6a and other vasohibins found in public databases. The branch is represented in red. Numbers next to the branches represent bootstrap values (in 1000 samplings). : Real time RT-PCR using total RNA samples from egg, miracidium, cercaria, schistosomulum or adult and primers for SmVASL. Relative fold change was calculated by comparing the Ct value for each sample to Ct values for alpha-tubulin (internal standard)

Analysis of genes shared with Deuterostomia and with possible roles in host interactions-5

<p><b>Copyright information:</b></p><p>Taken from "Analysis of genes shared with Deuterostomia and with possible roles in host interactions"</p><p>http://www.biomedcentral.com/1471-2164/8/407</p><p>BMC Genomics 2007;8():407-407.</p><p>Published online 8 Nov 2007</p><p>PMCID:PMC2194728.</p><p></p>ed yeast INSIG homologs were included in the MSA. SmINSIG transmembrane regions predicted by TMHMM and MINNOU are indicated by red and green bars, respectively. A consensus sequence and conservation bars are also represented; : Maximum Likelihood tree constructed from the alignment of SmINSIG and several INSIGs found in public databases. The branch is represented in red. Numbers next to the branches represent bootstrap values (in 1000 samplings)