Figure 3

Binding of C/EBPβ to proinflammatory gene promoters in activated mixed glial cultures. A. C/EBPβ DNA binding activity was analyzed by gel shift and supershift assays. Nuclear proteins were extracted from secondary mixed glial cultures treated with vehicle (lanes 1, 4, 7), LPS (lanes 2, 5, 8) or LPS+IFNγ (lanes 3, 6, 9) for 2 h. The first lane represents the probe without nuclear extract incubation (free probe). Arrows indicate four shifted complexes. Complex IV is a C/EBPβ independent complex. Lanes 1 to 3 show C/EBPs shifting complexes in wild type condition. Supershift with anti-C/EBPβ antibody (lanes 4 to 6) shows the presence of C/EBPβ in I-III complexes in all treatments. Rabbit IgG (lanes 7 to 9) is used as negative control for the supershift assay. This image is representative of four independent experiments. B. Quantitative analysis of C/EBPβ binding to NOS2, IL-1β, IL-6 and TNFα promoters by qChIP in mixed glial cultures. The sequences and positions of every C/EBPβ binding site and the primers used for qPCR are found in table 2. IL-10 was used as positive control. The qChIP assay was carried out after 2 h of LPS, LPS+IFNγ or vehicle (control) treatment. The IgG bars represent the means for IgG/Control, IgG/LPS and IgG/LPS+IFNγ PCR values for each gene. Input refers to total DNA. % of input represents the percentage of qChIP/Input ratio. One-way ANOVA, followed by Bonferroni's multiple comparison test is applied. **p < 0.01; ***p < 0.001 compared to control. #p < 0.05; ##p < 0.01; ###p < 0.001 compared to LPS. (n = 3)