Fractions were collected at the interface 20/30%, 30/40%, and 40/60%, diluted in 20 mM Tris-Hcl buffer pH 7.8, and pelleted (140,000 g for 30 min at

Selonsertib clinical trial 4°C). Pellets were resuspended in Tris buffer and loaded on SDS-PAGE. After staining protein bands were picked and washed and proteins were trypsinated. Peptides were analyzed by LC-MS/MS on an orbitrap. Transmission electron microscopy Sample preparation for ultrastructure Observations on whole trypanosomes obtained after incubation in secretion medium or directly from blood of infected rat were conducted as described previously [80]. After centrifugation, pellets of trypanosomes were fixed in 4% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.2) overnight at 4°C, washed in fixation buffer, postfixed in osmium tetroxide for 1 h at 4°C and washed. Pellets were dehydrated in an acetone series and embedded in TAAB 812 epon resin [80]. Thin sections, mounted on 75 mesh collodion carbon-coated copper grids, were contrasted with uranyl acetate and lead citrate and examined at 80 KV with a transmission electron microscope (Jeol 100CX II). Negative stain The stains were prepared according to Brun et al., 2008 [81]. Basically, 2-3 μl of sample (vesicles obtained by ultracentrifugation and sucrose gradient) were layered onto a 200-formvar-coated grids for

1 min. Liquid was remove with filter paper. The grid was incubated with a negative stain (1.5% uranylacetate in 70% ethanol for 1 min) and washed 3 times with water. The preparation was observed with a Hitachi H. 7100 transmission Tucidinostat electron microscope. Acknowledgements This work was funded by the

Foundation for Medical Research (FRM). We sincerely thank Dr Daniel Fisher for his kind proofreading of the manuscript. We thank Pr Philippe Vincendeau and Dr Philippe Holzmuller for providing secretion buffer and Dr Sophie Ravel for her technical help. We thank Dr Chantal Cazevieille from the CRIC (Centre Régional d’Imagerie Cellulaire) laboratory in Montpellier for the electronic Cyclin-dependent kinase 3 microscopy negative stain picture analysis, the Pôle Proteome de Montpellier, particularly Dr Serge Urbach and Martial Seveno for the MS/MS click here analysis of the TIRSP fraction on Orbitrap, and Dr Nicolas Sommerer for making complementary MS facilities available and for expert advice. Electronic supplementary material Additional file 1: Table S1. Secreted proteins identified in 3 T. brucei gambiense strains separated on 1D gel. contains the identification of the proteins secreted by Biyamina (sheet 1), Feo (sheet 2), and OK strain (sheet 3) and their classification according to functional categories (MapMan bins nomenclature). For each protein, the number of matched peptides and the highest score are described. (PDF 36 KB) Additional file 2: Table S2. Secreted proteins from OK strain identified on BN-PAGE gel.