Figure 3.

Inhibition of fimbria-stimulated NF-κB activation by melatonin and indole in RAW264.7 cells. Left hand panel: inhibitory effects of melatonin and indole on fimbria-stimulated NF-κB binding. The cells were pretreated for 30 min with or without the indicated doses of melatonin or indole and then incubated with or without fimbriae at 4 μg/mL for 1 h. A gel mobility shift assay was performed with nuclear proteins and ³²P-labeled oligonucleotide containing the NF-κB consensus sequence. Three independent experiments were performed, and similar results were obtained. Right hand panel: inhibitory effects of melatonin and indole on phosphorylation-dependent proteolysis of fimbria-stimulated IκB-α. The cells were pretreated for 30 min with or without melatonin or indole at 10 μM and then incubated with or without fimbriae at 4 μg/mL. Equal amounts of cell lysates were analyzed by Western Blotting after SDS-PAGE with phosphospecific anti-IκB-α antibody, anti-IκB-α antibody, or anti-β-actin antibody. Three independent experiments were performed, and similar results were obtained (Copyright In Vivo, Athens, Greece).