Effect of atRA on NF-κB/DNA binding. Chondrocytes were treated with or without tumour necrosis factor (TNF)-α (30 ng/ml) and/or all-trans retinoic acid (atRA; 1, 10, or 100 nmol/l) for 24 hours. Nuclear extracts were incubated with 32P-radiolabelled κB consensus DNA and resolved on a 4% polyacrylamide gel. TNF-α induced the formation of a complex of proteins that contained both nuclear factor-κB (NF-κB) p65 and retinoic acid receptor (RAR)α bound to the κB consensus site (a, lane 2). Addition of antibody against NF-κB p65 gave rise to a supershifted complex (b, lanes 3 and 11). Antibody against RARα interfered with binding of the complex to DNA (lanes 4 and 12). atRA decreased the amount of TNF-α-activated complex bound to the κB consensus site in a concentration-dependent manner (lanes 8 to 10). The TNF-α-activated complex that remained bound in the presence of atRA (0.1 μmol/l) contained p65 and RARα (lanes 11 and 12). Results shown are representative of three independent experiments.
Rockel et al. Arthritis Research & Therapy 2008 10:R3 doi:10.1186/ar2349