precleared using salmon sperm DNA/protein A-agarose and a sample of ‘‘input DNA’’ was collected at this point. Protein-DNA complexes were immunoprecipitated with 5 mg of Ab overnight at 4uC. Ab-protein-DNA complexes were then captured using salmon sperm DNA/protein A-agarose for 1 h at 4uC. After washing beads with low and high salt, LiCl, and TE buffers, the protein/DNA complexes were eluted using 1% SDS, 0.1 M NaHCO3 buffer and disrupted by heating at 65uC for 4 h. DNA was then extracted using phenol/chloroform extraction and ethanol precipitation. PCR was conducted using promoter specific primers: IL-10 promoter (STAT3 binding region): sense 59- TCATGCTGGGATCTGAGCTTCT-39, antisense 59-CGGAA- GTCACCTTAGCACTCAGT-39 (94uC, 15 s; 56uC, 30 s; 72uC, 1 min, 35cycles); IL-12p40 promoter (NF-kB binding site): sense 59-AGTATCTC TGCCTCCTTCCTT-39, antisense 59-GCAA- CACTGAAAACTAGTGTC-39 (initial denaturation at 95uC for 3 min; amplification cycles at 95uC for 30 s, 58uC for 1 min, and 72uC for 1 min; and a final extension at 72uC for 10 min, 40cycles). PCR amplified product was subsequently size fractioned on 2% agarose gel, stained with ethidium bromide and visualized under UV-light. For relative quantitation of promoter levels, real- time PCR was also performed. For real-time PCR samples were normalized to input DNA controls. Isolation of RNA and real-time polymerase chain reaction Total RNA extracted from macrophages (TRI reagent; Sigma) according to the standard protocol [44,45] was reverse transcribed using Revert Aid M-MuLV reverse transcriptase (Fermentas). Real-time polymerase chain reaction (PCR) was performed using SYBR Green mix and the ABI 7500 real-time PCR system (Applied Biosystems). Glyceraldehyde- 3-phosphate dehydroge- nase (GAPDH) was used as a reference. Sequences of the PCR primers are listed in Table 1. The reaction conditions consisted of an initial activation step (5 min at 95uC) and cycling step (denaturation for 30 s at 94uC, annealing for 30 s at 58uC, and extension for 1 min at 72uC for 40 cycles), after which melt curve analysis was performed. Detection of the dequenched probe, calculation of threshold cycles, and further analysis of these data were done using Sequence Detector software (version 1.4; Applied Biosystems). Relative changes in iNOS2 and cytokine messenger RNA (mRNA) expression were compared with unstimulated control, normalized to GAPDH, and quantified by the 2DDCt method. Isolation of RNA and RT-PCR RNA was isolated according to the standard protocol [44,45]. Briefly, total RNA extracted from macrophages (TRI reagent; Sigma) according to the standard protocol [44,45] was reverse transcribed using Revert Aid M-MuLV reverse transcriptase (Fermentas). The cDNA encoding the SOCS3, GAPDH gene was amplified using specific primers as listed in Table 1. PCR amplification was conducted in a reaction volume of 50 ml using a Perkin Elmer Gen Amp PCR system 2400 and 0.5 unit of Taq polymerase set for 35 cycles (denaturation: at 94uC for 30 s; annealing: at 58uC for 30 s; extension: at 72uC for 30 s) PCR References Ara-LAM Mediated Protection in Leishmaniasis Table 1. Sequences of the PCR primers. Gene iNOS2 IL-10 IL-12p40 TNF-a SOCS-3 GAPDH Sequence of Primers Forward 59-CCCTTCCGAAGTTTCTGGCAGCAGC-39 Reverse 59-GGCTGTCAGAGCCTCG TGGCTTTGG-39 Forward 59-CGGGAAGACAATAACTG-39 Reverse 59-CATTTCCGATAAGGCTTGG-39 Forward 59-CAACATCAAGAGCAGTAGCAG-39 Reverse 59TACTCCCAGCTGACCTCCAC-39 Forward 59-GGCAGGTCTACTTTGGAGTCATTGC-39 Reverse 59-ACATTCGAGGCTCCAGTGAATTCGG-39 Forward 59-GCGGGCACCTTTCTTATCC-39 Reverse 59-TCCCCGACTGGGTCTTGAC-39 Forward 59-CAAGGCTGTGGGCAAGGTCA-39 Reverse 59-AGGTGGAAGAGTGGGAGTTGCTG-39 doi:10.1371/journal.pone.0024141.t001 amplified product was subsequently size fractioned on 2% agarose gel, stained with ethidium bromide and visualizedunder UV-light. Densitometry analysis Immunoblots were analyzed using a model GS-700 Imaging Densitometer and Molecular Analyst (version 1.5; Bio-Rad Laboratories). Statistical analysis The in vitro cultures were performed in triplicate. The data, represented as mean values 6standard deviations, are from 1 experiment that was performed at least 3 times. Student’s t test was employed to assess the significance of the differences between the mean values of control and experimental groups. A P value of.05 was considered to be significant, and a P value ,.001 was considered to be highly significant. Ethics Statement This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experimental animal protocols received prior approval from the Institutional Animal Ethical Committee (Bose Institute, Registra- tion Number: 95/99/CPCSEA). Acknowledgments We are grateful to Director, Bose Institute (Kolkata) for his continuous encouragement. We cordially thank Mr. Debasish Majumdar and Mr. Prabal Gupta for their technical assistance. Author Contributions Conceived and designed the experiments: S. Majumdar PB SC. Performed the experiments: PB GG S. Majumder AA SB KH SBM. Analyzed the data: S. Majumdar PB. Contributed reagents/materials/analysis tools: S. Majumdar SC SR. Wrote the paper: S. Majumdar. 1. Descoteaux A, Matlashewski G (1989) c-fos and tumor necrosis factor gene expression in Leishmania donovani-infected macrophages. Mol Cell Biol 9: 5223–7. 2. Murray HW, Spitalny GL, Nathan CF (1985) Activation of mouse peritoneal macrophages in vitro and in vivo by interferon-gamma. J Immunol 134: 1619–1622. 3. Russell DG, Talamas-Rohana P (1989) Leishmania and the macrophage: a marriage of Inconvenience. Immunol Today 10: 328–33. 4. Murray HW (1982) Cell-mediated immune response in experimental visceral leishmaniasis. II. Oxygen-dependent killing of intracellular Leishmania dono- vani amastigotes. J Immunol 129: 351–7. PLoS ONE | www.plosone.org 11 September 2011 | Volume 6 | Issue 9 | e24141