Af519

Hamster BHK-21 cell line was transiently transduced with lentiviral particles carrying a STAT-3 luciferase reporter construct (Cignal Lenti Reporter, Qiagen). Cells (5,000 cells/100 μL) were transduced at 20 MOI in 8 μg/mL polybrene in DMEM with 10% FBS. At 16 hrs post-transduction the medium was changed, and after 72h cells were exposed or not to L. donovani promastigotes (1:5), treated with or without recombinant hamster IFN-γ, and pre-treated with or without 2 μg/mL of anti-IL-10 antibody (mouse/rat anti-IL-10, R&D, AF519, R&D). Activity of the STAT-3 reporter was expressed as relative luciferase activity per number of cells determined with cell titer Glo (Promega).

Mice were killed with overdoses of urethane, and the L4–L6 spinal cord segment was rapidly removed. The dorsal horn tissues were homogenized in lysis buffer containing a mixture of protease inhibitors and phenylmethylsulfonyl fluoride (Roche Diagnostics). Equal amount of protein was loaded and separated in 10% Tris-Tricine SDS-PAGE gel and transferred to PVDF membrane (Millipore). The membranes were blocked with 5% nonfat milk in Tris-buffered saline (pH 7.5) with 0.1% Tween-20 for 2 h at room temperature (RT) and incubated overnight at 4°C with goat anti-IL-10 antibody (1 : 250, R&D, AF519, USA), and goat anti-IL-10RA antibody (1 : 1000, R&D, AF-474-NA, USA). The blots were then incubated with HRP-conjugated secondary antibodies (1 : 2000, Pierce) for 2 h at 4°C. GAPDH antibody was probed as a loading control. Signals were finally detected using enhanced chemiluminescence (ECL, Thermo, USA), and the bands were visualized with the ChemiDoc XRS system (Bio-Rad, USA). All Western blot analysis was performed at least three times, and consistent results were obtained.