Ara-LAM Mediated Protection in Leishmaniasis Figure 3. Histone H3 modifications at the IL-10 promoter in Ara-LAM treated infected macrophages. Murine macrophages (16106 cells/ mL) were treated with SB or PD for 2 h, subsequently followed by Ara-LAM treatment for 3 hr and Leishmania infection for 45 min. After 45 min of incubation, ChIP assays were conducted as described in Materials and Methods. Immunoprecipitations were performed using Abs specific to phosphorylated H3 (IP phospho-H3) (A) or IP acetyl-H3 (B), and conventional RTPCR or quantitative real-time PCR was performed using primers specific to the IL-10 promoter. *P,.001 compared with Ara-LAM–pretreated infected macrophages. In a separate experiment, the macrophages were treated as described above followed by infection with Leishmania parasite. After 24 hr of incubation, the cells were lysed and subjected to Western blot with anti- pSTAT3, and STAT3 as described in Materials and Methods (C). Peritoneal macrophages (36106 (cells/mL) were treated and infected by Leishmania for 15, 30 min as described in the legends of figure 1. Cytosolic and nuclear protein extracts were prepared for Western blot analysis as described in material method to analyze the nuclear translocation of pSTAT3 and STAT3 (D). The blot shown is a representative of experiments performed in triplicate. Band intensities were analyzed by densitometry (i,ii). (inset). In a separate experiment murine macrophages (16106 cells/mL) were treated and infected with Leishmania parasite as described above. After 45 min of incubation, Immunoprecipitations were conducted using STAT3 (IP: STAT3) specific Abs. Conventional RT-PCR or quantitative real-time PCR was performed for amplifying the putative STAT3 binding sites of the IL-10 promoter. *P,.001 compared with Ara-LAM–pretreated infected macrophages. doi:10.1371/journal.pone.0024141.g003 PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24141