Fig 1. Ara-LAM enhanced IFN-γ Receptor alpha chain expression in parasitized macrophages.

(A) BALB/c derived peritoneal macrophages (2x10⁶cells) were infected with Leishmania donovani (cell: parasite 1:10) for 30mins, 1hr, 3hrs, 12hrs, 18hrs and 24hrs. Other sets of macrophages were pre-treated with Ara-LAM for 3 hrs and followed by infection for the same time periods. Cell lysates were prepared followed by Western blot for IFN-γRα. Data are from 1 of 3 experiments conducted in the same way with similar results. IFN-γRα expression in Ara-LAM treated infected macrophages were significantly higher than the infected cells (p<0.05). (B) In a separate set of experiment, BALB/c derived peritoneal macrophages (2x10⁶cells) were pre-treated with Ara-LAM for 3 hr and followed by Leishmania donovani infection for 24 hr. Macrophages were then treated with rIFN-γ (20ng/ml) for 45min, followed by cell lysate preparation and Western blotting for IFN-γRα. The blots shown are representative of three experiments. (C) Ara-LAM pre-treated peritoneal macrophages (2x10⁶cells) were infected with L. donovani for 24 hrs followed by rIFN-γ (20ng/ml) stimulation for another 24 hrs, and staining with IFN-γRα-PE Ab and analyzed on a flow cytometer. Data are from one representative experiment that was repeated thrice. (D) Treated and untreated infected macrophages (2x10⁶cells) were subjected to immuno-precipitation using NF-κB (IP:NF-κB) specific Ab. Semi quantitative RT-PCR was performed for amplifying the putative NF-κB binding sites at the IFN-γRα promoter. Data are from one representative experiment, which was performed at least thrice.