Figure 7. Inhibition of LPS-induced NO production in exopro- teome-treated macrophages. B10R macrophages were untreated (Nil), infected with L. mexicana or incubated with exoproteome for 16 h and stimulated with 10 or 100 ng/ml of LPS for 20–24 h the following day. Neither L. mexicana infection nor exoproteome induce noticeable amounts of NO. Furthermore, L. mexicana infection hampers NO production by 70 and 80% after 10 and 100 ng/ml of LPS stimulation respectively. Exoproteome incubation hampers NO production by 50 and 30% after 10 and 100 ng/ml of LPS stimulation respectively (p- value,0.0001). doi:10.1371/journal.pone.0018724.g007 Prediction of nonconventionally secreted proteins was performed using SecretomeP 2.0 server [31]. Scanning Electron Microscopy Parasites were fixed in 2.5% glutaraldehyde fixative solution overnight. The following day, samples were put on poly-L-lysine coated slides and dehydrated in ethanol, amyl acetate and supercritical CO2 sequentially. Dehydrated samples were coated for 3 min with Au-Pd and visualized using a Hitachi S-4700 Cold Field Emission Gun Scanning Electron Microscope (FEGSEM). Ultracentrifugation Ultracentrifugation for sedimentation of exovesicles from the exoproteome was done according to standard methods [22]. Briefly, exovesicles were prepared by centrifugation of the exoproteome at 10,0006 g for 35 min, to clear possible cell debris followed by 1 h centrifugation at 100,0006 g. Pellet was resuspended in PBS and stored at 280uC or fixed immediately with glutaraldehyde 2.5% for visualization by SEM. In vitro infection B10R macrophages were infected with stationary L. mexicana parasites at 1:20 ratio for 3 or 16 h. Macrophages were similarly incubated with ,25-fold concentrated and 0.22 mm filter- sterilized exoproteome for 3 or 16 h. Following incubation or infection, cells were washed with PBS and lysed. In gel PTP assay In gel PTP assay were performed as described previously [40]. Briefly, poly (Glu-Tyr) substrate was radiophosphorylated with FER protein kinase and 150 mCi of [c-32P] deoxyadenosine 59-triphos- phate. Substrate was precipitated by TCA and then incorporated in an SDS-polyacrylamide gel at a concentration of 26105 cpm/ml. Cell lysates were run on SDS-PAGE and then gels were incubated for 20 h in 50 mM Tris-HCl (pH 8.0) and 20% isopropanol and washed twice in 50 mM Tris-HCl (pH 8.0), 0.3% b-mercaptoethanol (b- Secreted Proteins of Leishmania Alter Macrophage ME). Gels were put in 6 M guanidine hydrochloride and 1 mM EDTA denaturation solution for 3 h, and then washed twice in 50 mM Tris-HCl (pH 8.0), 1 mM EDTA, 0.3% b-ME and 0.04% Tween 20, renaturation buffer. Final renaturation was done overnight and the gels were dried and autoradiography performed using Kodak film. Clear bands were indicative of active PTPs. pNPP assay pNPP assay was performed as described previously [3]. Protein concentration of cell lysates was measured by Bradford assay and equal amounts of protein (10–15 mg) were incubated in a phosphatase reaction mix containing 50 mM HEPES (pH 7.5), 0.1% b-ME and 10 mM pNPP (Fluka) for several minutes at 37uC. pNPP hydrolysis was quantified spectropscopically at 405 nm. pNPP assay of exoproteome was performed similarly by incubating equal volumes of acquired exoproteome from indicated conditions with the reaction mix. Western blot Western blot analysis of cell lysates and precipitated exopro- teome was performed according to standard methods. Proteins were electrotransfered to Hy-bond nylon membrane (GE Health) and were detected with primary antibodies against peroxiredoxin (Lashitew Gedamu, University of Calgary, Canada), HSP70 (Jose´ Requena, University Autonoma de Madrid, Spain), HSP83 (Greg Matlashewski, McGill University, Canada), CPB (Jeremy Mot- tram, University of Glascow, UK), gp63 (Robert McMaster, University of British Columbia, Canada), LACK (Jean-Claude Antoine, Institut Pasteur, France), SHP-1 (Chemicon, CA), and PTP-1B (Upstate), HGPRT and hexokinase (generated in our laboratories). Anti-mouse or anti-rabbit antibodies conjugated to horse-radish peroxidise (HRP) (GE Health) were used as secondary antibodies. Membranes were developed with the ECL Western blot detection system (GE Health). Electromobility shift assay (EMSA) EMSA was performed as described previously [3]. Briefly, nuclear proteins were extracted using an isotonic and then a hypotonic buffer. Extracted nuclear proteins were incubated with radiolabelled consensus sequences of NF-kB (59-AGTTGAGGG- GACTTTCCCAGGC-39), AP-1(59-AGCTCGCGTGACTCAG- CTG-39) and SP-1 (59-ATTCGATCGGGGCGGGGCGAGC- 39) (Santa Cruz) as non-specific control. Samples were run on a native 4% acrylamide gel. Following electrophoresis, gels were dried and autoradiography was performed. Nitric oxide assay Nitric oxide (NO) assay was performed as described previously [3]. Briefly, B10R macrophages were infected or incubated with exoproteome for 16 h. The following day, parasites and supernatant were washed out and cells were incubated for 24 h with 10 or 100 ng/ml of LPS in phenol red-free RPMI. Concentration of nitrite was measured using Greiss reaction. Image analyses Densitometry and particle size measurements were done using ImageJ 1.42I software from National Institutes of Health. Statistical analyses Statistical analyses were performed by Graphpad Prism 5.0 using an unpaired t-test. All results are representatives of at least 3 independent experiments. PLoS ONE | www.plosone.org 8 May 2011 | Volume 6 | Issue 5 | e18724