Figure 7.

Effect of nepalolide A on the electrophoretic mobility shift assay (EMSA) for NF-κB and AP-1 binding. C6 glioma cells and astrocytes were preincubated with nepalolide A (2–10 μM) for 30 min. After 30 min incubation, cells were challenged with 5 μg ml⁻¹ LPS/100 units ml⁻¹ IFN-γ (LPS/IFN-γ) or 5 ng ml⁻¹ TNF-α/5 ng ml⁻¹ IL-1β/100 units ml⁻¹ IFN-γ (mixed cytokines) plus or minus nepalolide A for 3 h. After incubation, nuclear extracts were isolated. The EMSA of NF-κB and AP-1 binding was performed by using 5′-end-labelled consensus or mutant oligonucleotides. (a and b) are representative autoradiographs of NF-κB and AP-1 binding, respectively. (c) is the relative level of NF-κB binding in untreated cells and cells treated with nepalolide A. Results are means±s.d. (where large enough to be shown) from five independent experiments, and are expressed relative to cells treated with iNOS inducers alone. Significant differences between control and nepalolide A-treated cells are indicated by ^**P<0.01; and ^***P<0.001.