Figure 3. Time-course of TNF--induced NF-B-specific DNA-protein complex formation and NF-B translocation in synovial fibroblasts from TMJ. Cells were treated with 20 ng/mL TNF- for various time intervals, and then cytoplasmic and nuclear extracts were prepared. (A) NF-B-specific DNA-protein-binding activity in nuclear extracts was determined by EMSA. (B) Supershift assays were performed with specific p65 and p50 antibodies. Moreover, we added unlabeled NF-B probes in a 100-fold excess, to determine the specificity of NF-B-specific DNA protein. (C) Cytoplasmic and nuclear levels of p65 and p50 proteins were detected by Western blotting. The data represent 1 of 3 separate experiments with similar results.