Secreted Proteins of Leishmania Alter Macrophage Figure 5. Exoproteome of L. mexicana induces modulation of macrophage PTPs, specifically SHP-1 and PTP1B. B10R macrophages were untreated (Nil), infected with L. mexicana parasites or incubated with exoproteome for 3 h. (A) Cleavage and activation of SHP-1 and PTP-1B as observed by western blot. (B) In-gel assay shows modulation of PTPs. Multiple active PTP bands appear following infection or exoproteome treatment (C) Augmentation of PTP activity following infection or exoproteome treatment can be further observed by phosphate analog pNPP assay (p- value,0.0001). C. f. denotes cleavage fragment. MW denotes molecular weight. doi:10.1371/journal.pone.0018724.g005 Materials and Methods Cell and Parasite culture Immortalized B10R bone-marrow derived macrophages derived from B10A.Bcgr mice were obtained from the laboratory of Dr. Danuta Radzioch (McGill University, Canada) and were cultured as described previously [32]. Briefly, cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco-BRL) supplemented with 10% heat-inactivated fetal bovine serum (FBS), streptomycin (100 mg/ml), penicillin (100 U/ml), and 2 mM L-glutamine at 37uC and 5% CO2. L. mexicana (MNYC/BZ/62/M379) parasites were cultured by bi-weekly passages at 26uC in Schneider’s Drosophila Medium supplemented with 10% FBS. Flow cytometry Measurement of cell viability was performed according to standard protocols [33]. Briefly, parasites were stained with propidium iodide (PI) and analyzed with FACS Calibur (BD Biosciences). Acquired data was analyzed by FlowJo. ml in serum free DMEM or phenol red-free RPMI media and incubated for 2–4 h. Culture supernatants were isolated by centrifugation twice at 4000 rpm for 10 min. Proteins in the supernatant were dosed using Quick start Bradford reagent (Biorad), precipitated with 15% trichloroacetic acid (TCA)/ acetone or concentrated ,25-fold using 10KD-cut off centrifugal column (Amicon Ultra). Tryptic digestion Gel extraction Proteins from gel bands (5 to 7 gel pieces per band/well) were subjected to reduction, cysteine-alkylation, and in-gel tryptic digestion by using an automated MassPrep workstation (Micro- mass), as previously described [34]. In solution digestion of TCA/ acetone precipitated samples was performed by the addition of trypsin (Promega) at a ratio of 1:25 (w/w) protease:protein. After an overnight incubation at 37uC, the reaction was quenched by the addition of formic acid to a final concentration of 1%. Samples were then cleaned using Zip Tip C18 before mass spectrometry analysis. Exoproteome preparation and proteomic analysis Stationary L. mexicana promastigotes were washed 3 times in phosphate buffer saline (PBS) and resuspended at ,108 parasites/ Liquid chromatography-mass spectrometry (LC/MS/MS) Extracted peptides were injected onto a Zorbax C18 (Agilent) desalting column and subsequently chromatographically separated PLoS ONE | www.plosone.org 6 May 2011 | Volume 6 | Issue 5 | e18724