Figure 4

Kinetics of TNF-α–induced nuclear factor-κB (NF-κB)-DNA binding activity in chondrocyte nuclear extracts. A, NF-κB-DNA binding activity was determined in chondrocyte nuclear extracts by EMSA. Lane 1, control; lane 2, TNF-α (10 ng/ml, 1 hour); lane 3, Bay 11-7085 (20 μm, 1 hour); lane 4, Bay 11-7085 (1 hour) followed by TNF-α (1 hour); lane 5, SNP (1 mm, 2 hours). The arrow indicates the p65/50 complex. B, NF-κB activity from chondrocyte nuclear extracts after TNF-α (10 ng/ml) stimulation for the indicated times. The first and the last lanes show NF-κB-DNA binding activity in nuclear extracts from nontreated chondrocytes at the beginning and the end of the experiment, respectively. C, NF-κB activity in chondrocyte nuclear extracts pretreated with TNF-α (10 ng/ml) for 48 hours and then treated with SNP (1 mm) for an additional 10 hours. Lane 1, control; lane 2, TNF-α; lane 3, SNP (1 mm); lane 4, TNF-α followed by SNP. D, Supershift analysis. Nuclear extracts from unstimulated (lanes 1–3) and TNF-α–treated (lanes 4–6) chondrocytes were analyzed for DNA binding to a κB probe either in the absence of antibody (lanes 1 and 4) or in the presence of an anti-p65 (lanes 2 and 5) or an anti-p50 antibody (lanes 3 and 6).