In parallel, cell lines were infected with VSVG-pseudotyped lentiviral particles as a positive control

r a4/9. Inside this novel group of platyhelminth-specific a-integrins, and in accordance with the presence of three to four a-integrins in each species, the receptors clustered in four families, which we called platyhelminth a1-, a2-, a3-, and a4-families. In a phylogenetic analysis with different integrin b receptors, the S. mansoni homolog clustered with orthologs from S. haematobium, S. japonicum, C. sinensis, E. multilocularis, and S. mediterranea. The only non-platyhelminth integrin receptor clustering with this group was the human integrin b4. Interestingly, it 21990348 was previously shown that this receptor is the only vertebrate integrin ortholog clustering outside of the b1 and b3 integrin families. Our phylogenetic analysis suggests that human b4 may have an evolutionary ancient origin, clustering in an own group close to the platyhelminth orthologs. Since this group was also 18519091 found to be unique, it was named platyhelminth bfamily. Within the platyhelminth a-integrin families and the platyhelminth b-family the phylogenetic relationship of platyhelminthes is perfectly reflected. surrounding area anterior the ovary, but not within the ovary. No signals were detected using sense probes of SmbInt1, Sma-Int1, or Sma-Int2 indicating that antisense regulation may not occur for these integrins. However, in situ hybridizations with probes specific for Sma-Int3 or Sma-Int4 led to different results. Antisense as well as sense transcripts of Sma-Int3 as well as Sma-Int4 were detected in the ovary indicating that the expression of these two aintegrins might be post-transcriptionally regulated by antisense RNA, a phenomenon increasingly reported for some schistosome genes including those involved in signal transduction. Interaction Studies in the Yeast Two-hybrid System and Co-immunoprecipitation Confirmed Smb-Int1-CTK Interactions Integrins are discussed to transduce signals via their intracellular part to signaling molecules such as Src and Syk CTKs. Since evidence has been obtained for a Src-Syk kinase complex in the reproductive organs of schistosomes, we performed interaction studies in the YTH system to investigate the potential of Smb-Int1 to be involved as an upstream partner. To this end the intracellular C-term of Smb-Int1 was expressed in yeast as a fusion protein with the Gal4-AD, and also the N-term protein interaction parts of schistosome CTKs were co-expressed as fusion proteins with the Gal4BD. All yeast clones survived growth selection, but b-Gal colony lift filter-assays showed LacZ expression exclusively for yeast clones which were transformed with Smb-Int1-C-term together with the SH4SH3 domains of SmTK3 and SmTK6, respectively, as well as with SmTK4-SH2SH2, indicating interactions of all three CTKs with the intracellular part of Smb-Int1. To quantify and compare the strengths of interactions, bGal liquid assays were performed. In all cases the interactions were confirmed, although the strongest interaction occurred Mertansine site between the SmTK4 SH2SH2-domain and Smb-Int1. To confirm the strong interaction between Smb-Int1 and SmTK4, a co-immunoprecipitation experiment was performed. To this end, we co-expressed in Xenopus oocytes the HA-tagged intracellular part of Smb-Int1 and the V5-tagged SmTK4 kinase. Oocyte lysate proteins were immunoprecipitated with each antitag antibody. The results showed the presence of V5-SmTK4 in the immune complexes isolated with anti-HA antibodies and, inversely, the presence of HA-Smb-Int1 in the V5 immune co