Electrophoretic Mobility Shift Assay for NF-κB

Extracts were prepared as previously described [16 (link)]. The protein amount was determined by the Bradford colorimetric procedure (Bio-Rad, Marnes-la-Coquette, France). For electrophoretic mobility shift assay (EMSA), 5 fentomoles of biotinylated probe (corresponding to consensus NFkB binding site) and 5 μg of nuclear proteins from chondrocytes were incubated at room temperature for 30 min in binding buffer (50 mM Tris–HCl pH 8, 1 mM dithiothreitol, 750 mM KCl, 2.5 mM EDTA, 0.5% Triton-X100; 65.5% glycerol (v/v)) with poly-dIdC (1 μg). Then all samples were loaded on a nondenaturating acrylamide gel (7%). After separation, probes and proteins were electrotransferred on PVDF membrane, which was incubated with streptavidin-conjugated peroxydase (Sigma). The signals were revealed with SuperSignal West Pico Chemiluminescent Substrate (Pierce Perbio Science, Brébières, France) and exposed to X-ray film.