The membrane was then incubated with rabbit polyclonal iNOS antibody (Sigma) followed by anti-rabbit immunoglobulin-horse radish peroxidase (Ig-HRP) conjugate (Sigma-Aldrich). Bound enzyme was detected by chemiluminescence following the manufacturer’s protocol (GE Healthcare, Piscataway, NJ). RAW 264·7 macrophages were seeded at a density of 5 × 106 per well in a six-well culture

plate and either left untreated or pretreated with PDTC for 1 hr, followed by stimulation with 5 μg of rRv2626c alone or with a combination of LPS and ΙFN-γ. Cells were harvested and nuclear extract was prepared from NP-40 lysed cells.36 Equal amounts of the protein extracts (50 μg) were fractionated on a 10% SDS-PAGE gel. The nuclear proteins were transferred onto a nitrocellulose membrane and incubated with polyclonal BMN-673 rabbit antibody to NF-κB p50 or NF-κB p65 (Santa Dabrafenib cell line Cruz Biotech, Santa Cruz, CA) followed by incubation with anti-rabbit Ig-HRP conjugate. Bound enzyme was detected by chemiluminescence (ECL). An equal amount of the nuclear extract (10 μg)

from each set (cells stimulated with rRv2626c, or rRv2626c + LPS or rRv2626c + IFN) was incubated at 37° for 30 min with 1 ng of γ-P32-radiolabelled consensus oligodeoxyribonucleotides containing the binding site for NF-κB (5′-ttgttacaagggactttccgctggggactttccagggaggcgtgg-3′; Santa Cruz Biotech) in a binding buffer [10 mm Tris, pH 7·5, 50 mm NaCl, 1 mm ethylenediaminetetraacetic acid (EDTA), 10% glycerol, 1 μg of poly dIdC, 1 mm dithiothreitol (DTT), 1 mm phenylmethylsulphonyl fluoride (PMSF) and 50 mm MgCl2]. For competition experiments, 100-fold molar excess of unlabelled consensus NF-κB or mutant NF-κB oligos was used to check the specificity of the DNA–protein complex. The DNA–protein complexes were resolved by electrophoresis on a 7% native PAGE gel at PAK6 4° in 1× Tris-borate-EDTA

(TBE). After electrophoresis, the gel was dried and exposed to Phosphor Imager screen (Fuji Film, Tokyo, Japan) at room temperature for 12 hr and the screen was scanned using the Typhoon system (GE Healthcare, Piscataway, NJ). Patients with TB who participated in this study were diagnosed at the Mahaveer Hospital and Research Centre, Hyderabad, India; their TB was confirmed by a tuberculin skin test, radiographic examination, and observation of acid-fast bacilli in sputum. Healthy controls were volunteers at the Centre for DNA Fingerprinting and Diagnostics who had no clinical symptoms of TB disease. Blood samples (2–3 ml) were collected from patients with TB (n = 48) as well as from healthy controls (n = 9), followed by separation of PBMCs on Ficoll-Histopaque (Sigma-Aldrich) as described previously.38 PBMCs were plated at a density of 2 × 105 per well in a 96-well culture plate and treated with rRv2626c (5 μg/ml) for 72 hr.