Figure 3

Effect of TNF-α on Sox9-DNA binding and Sox9 nuclear protein levels. (a) Chondrocytes were treated for 24 hours with or without tumour necrosis factor (TNF)-α (30 ng/ml). Nuclear extracts (10 μg) were incubated with double-stranded ³²P-labelled oligonucleotides corresponding to the Col2a1 minimal enhancer sequence and resolved on a 4% polyacrylamide gel. Where indicated, excess unlabelled specific oligonucleotides (a: 40× or b: 80×) were added as competitors. TNF-α did not change the amount of protein complex (arrowhead) bound to the oligonucleotide. Results shown are representative of three independent experiments. (b) Chondrocytes were treated for 24 hours with or without TNF-α (30 ng/ml) and/or atRA (100 nmol/l). Nuclear extracts (30 μg) were resolved on a 7.5% polyacrylamide gel and immunoblotted with antibody recognizing Sox9. The 64 kDa band corresponding to Sox9 (arrowhead) was quantified by densitometry. Data were normalized as a fraction of band density in untreated chondrocytes and are expressed as means ± standard error (three independent experiments). Data were evaluated by one-way analysis of variance. There was no significant change in the level of Sox9 nuclear protein (P > 0.05).