Mouse anti human beta actin

Mouse anti-human beta-actin, rapamycin and doxycycline hyclate were from Sigma. Helenalin, SN50 and Compound C were from Merck Biochemicals. Caffeic acid phenethyl ester (CAPE) was from Tocris (R&D Biosystems). Mouse anti-human MMP-8, mouse anti-human MMP-9, rabbit anti-human GAPDH, rabbit anti-human histone 2B, rabbit anti-human phospho-p70S6k (T229), rabbit anti-Mycobacterium tuberculosis, rabbit anti-human neutrophil elastase, rabbit anti-human phospho-AMPK alpha 1 and 2 (T172), sheep anti-human histone 2B, rabbit anti-human histone H3 (citrulline 2 + 8 + 17), donkey anti-Sheep IgG DyLight 488, goat anti-mouse DyLight 549, goat anti-rabbit IgG Cy5 were from Abcam. Rabbit anti-human phospho-Akt, total-Akt, phospho-AMPKα1/2 (T172), total AMPKα, and goat anti-rabbit HRP linked were from Cell Signalling Technology. Goat anti-mouse IgG (H+L) was from Jackson Immunoresearch laboratories. Goat anti-human MMP-8 was from R&D Biosystems and mouse anti-human MMP-9 was from Millipore. Rabbit anti-human MMP-8 was from Novus Biologicals. Mouse anti-human neutrophil elastase was from Dako. mouse anti-human MMP-9 was from Millipore.

Cell lysates were separated using SDS-polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes. Membranes were blocked in Tris-buffered saline (TBS; 0.05 M Tris-HCl and 0.2 M NaCl, pH 7.4) containing 0.1% (v/v) Tween-20 (TBST) and 5% non-fat milk (w/v) for 1 hr at room temperature then probed with the appropriate primary antibody; either mouse anti-human IDO (RayBiotech, Norcross, GA, USA) or mouse anti-human beta-actin (Sigma-Aldrich, St Louis, MO, USA) diluted in TBST containing 5% non-fat milk. Probing was performed overnight at 4°C. After rinsing the membranes were incubated with HRP-conjugated anti-mouse IgG for 1 hr at room temperature, and then rinsed again. Band size was determined by comparison with a biotinylated protein ladder (Cell Signaling Technology Inc., Danvers, MA) and the density of each band was determined using ImageJ image analysis software [43 ] using a method detailed by Miller [44 ]. Results are reported as the relative abundance: a ratio of the density of the IDO band to the beta-actin band.