Figure 3.
Azithromycin (AZM) inhibited the association of activator protein (AP)-1 and interferon consensus sequence binding protein (ICSBP)/ nuclear factor of activated T-cells (NFAT) with DNA-binding sites in the IL-12p40 promoter. Effect of AZM on AP-1 binding activity for the IL-12p40 promoter was assessed by EMSA. Nuclear extracts were obtained from resting RAW264.7 cells (lane 1), cells pre-treated with vehicle followed by stimulation with LPS/IFN-γ (lane 2), and from cells pre-treated with AZM (20 μg/mL) followed by stimulation with LPS/IFN-γ (lane 3). In lane 4, nuclear extract from cells pre-treated with vehicle followed by stimulation with LPS/IFN-γ were treated with excess cold probe. AP-1-DNA binding activity was enhanced by LPS/IFN-γ stimulation. Enhanced binding was inhibited by pre-treatment with AZM. Specific competition with cold probe eliminated the DNA-nuclear protein complex band. Effect of AZM on ICSBP/NFAT binding activity for IL-12p40 promoter was assessed by EMSA. Nuclear extracts were obtained from resting RAW264.7 cells (lane 5), cells pre-treated with vehicle followed by stimulation with LPS/IFN-γ (lane 6), and from cells pre-treated with AZM (20 μg/mL) followed by stimulation with LPS/IFN-γ (lane 7). In lane 8, nuclear extract from cells pre-treated with vehicle followed by stimulation with LPS/IFN-γ were treated with excess cold probe. ICSBP/NFAT DNA binding activity was enhanced by LPS/IFN-γ stimulation. Enhanced binding was inhibited by pre-treatment with AZM. Specific competition with cold probe eliminated the DNA-nuclear protein complex band. Representative images from three independent experiments are shown. “+” indicates the presence of pretreatment of AZM, stimulation of LPS/IFN-γ or cold probe in experiment of each lane. “-” indicates the absence of pretreatment of AZM, stimulation of LPS/IFN-γ or cold probe in experiment of each lane.