Figure 6.

Egr-1 DNA binding activity is increased by TNFα-induced MEK1/2 signalling in chondrocytes. Cells were pretreated with dimethyl sulfoxide, U0124 (10 μM) or U0126 (10 μM) for 30 minutes prior to treatment with vehicle (-) or with 30 ng/ml TNFα (+) for 90 minutes. Nuclear extracts were incubated with ³²P-radiolabelled oligodeoxynucleotides corresponding to the Egr consensus DNA binding sequence. In some cases, the nuclear extracts were incubated with 100-fold excess of cold specific Egr consensus oligodeoxynucleotides (egr), mutant Egr oligodeoxynucleotides (mut) or nonspecific oligodeoxynucleotides corresponding to the NFκB consensus sequence (κB). For antibody interference assays, nuclear extracts were preincubated with specific antibody for Egr-1 (egr) or nonspecific antibody for NFκB p65 isoform (p65). Resulting protein-DNA complexes were resolved on 4% polyacrylamide gels and exposed by autoradiography. Arrows, Egr-1-containing complexes. The autoradiograph shown is representative of three independent experiments.