The supernatant was recovered and conserved at ?80��C. The total protein amount present in the final sporocyst sample was determined with 2-D Quant Axitinib Kit (Amersham Biosciences). Plasma protein recovery Hemolymph of two hundred Brazilian B. glabrata snails (BgBRA) (9�C13 mm) was extracted as previously described [32]. It represents a total volume of 20ml approximately. A centrifugation (3000g; 10min; 4��C) was performed to pellet hemocytes and the plasma recovered (supernatant). Then, haemoglobin was removed from plasma using an ultra-centrifugation procedure (55 000 rpm; 2.5 hours; 4��C). Quantification of total protein concentration was performed with the 2-D Quant Kit (Amersham Bioscience). Plasmas were conserved at ?80��C. S. mansoni/B.

glabrata interactome experiments Fifty ��g of sporocyst extracts from C or IC strain and 750��g of plasma extracts were used for each interactome experiment. After thawing, extracts were submitted to a centrifugation step of 7 500g for 30 min at 4��C. The supernatants were recovered, mixed and incubated at 26��C for 2.5 hours. After incubation precipitated materials were recovered by two successive centrifugation steps at 7 500g and 15 000g for 30 min and at 4��C. The same procedure was realised with sporocyst and plasma extracts alone to identify proteins precipitating spontaneously. Precipitated proteins were resuspended in 7��l of UTCD (8M urea, 40 mM TRIS, 4% CHAPS, 60 mM DTT), 3��l of laemmli buffer 3�� was added and precipitates were analysed by SDS-PAGE. Gels were silver stained using a staining procedure compatible with mass spectrometry analysis [33].

Production and purification of recombinant SmPoMuc and co-immunoprecipitation Construction of expression vector and production of recombinant SmPoMuc1 The last 699 bp sequence of SmPoMuc1 (GenBank accession number: “type”:”entrez-nucleotide”,”attrs”:”text”:”EU042599″,”term_id”:”156118914″,”term_text”:”EU042599″EU042599) encoding the constant C-terminal region (from amino acid 199 to amino acid 432) was amplified and cloned into the NheI/SacI sites of the pET200/D-TOPO expression vector in frame with a hexahistidine tag (Invitrogen). Briefly, the 699 bp cDNA fragment of SmPoMuc1 (rSmPoMuc) was obtained using a standard amplification reaction with the following primers containing NheI or SacI restriction sites (5�� primer : CTA-CTA-CTA-gct-agc-GTT-CCA-GAA-CAT-TTG-AAA-ACG-A and 3�� primer ATT-ATT-ACA-gag-ctc-ATC-AGC-TGC-AAT-TGG-TTG-AAT-CTT).

Transformation of the plasmid construct was done in TOP10 chemically competent E. coli cells (Invitrogen) and sequencing was performed using T7 forward and reverse primers to verify its open reading frame. For production of rSmPoMuc-tagged protein, plasmid construct was transformed into Bl21 (DE3) E. coli competent cells. Transformed bacteria were grown in LB broth medium Entinostat with kanamycin (50��g/ml) at 28��C.