Bradford colorimetric procedure

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.

After treatment, sponges containing cells were rinsed once with ice-cold PBS, crushed and total proteins were extracted from cells using the RIPA-lysis buffer with a protease inhibitor cocktail. Protein concentration was assessed according to the Bradford colorimetric procedure (Bio-Rad SA). Then, 20 μg of total proteins were separated in 10% polyacrylamide gels containing 0.1% SDS and transferred to a polyvinylidene difluoride membrane (PVDF, Millipore). Unspecific binding sites of the membrane were blocked with 10% non-fat milk powder in Tris-buffered saline with 0.1% Tween (TBST) for 2 h. Then, membranes were incubated overnight at 4 °C with rabbit anti-human type I collagen (Novotec), rabbit anti-human type II collagen (Novotec) or rabbit anti-human GAPDH (Santa Cruz Biotechnology, Inc.). The following day, membranes were washed three times, followed by an incubation with HRP-conjugated goat anti-rabbit IgG antibody (Jackson Immunoresearch). Signals were visualized with the chemiluminescence method (ECL plus western blotting detection reagent+, Santa Cruz Biotechnology, Inc.) and developed on X-ray film.