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Fig. 5 | Journal of Neuroinflammation

Fig. 5

From: TLR4-RelA-miR-30a signal pathway regulates Th17 differentiation during experimental autoimmune encephalomyelitis development

Fig. 5

RelA binds to specific sites in the regulatory elements of miR-30a gene. a An expanded view of a 6-kb region (− 5000 bp~1000 bp of miR-30a gene) centered on the homologous binding sites of transcription factors among the species of mouse, human, and chimp. The region is divided into 7 parts (I–VII) according to the binding sites. Peaks represent the level of homology. b Binding site deletion analyses in the 5-kb up-stream region of miR-30a gene using a luciferase assay. On the left side is a schematic representation of the deleted DNA sequences carrying different clusters. The right panel shows luciferase activity normalized to Renilla luciferase activity. The construct carrying all clusters is used as control. c RelA binding sites in the 6-kb region. Red box shows the concentration of binding sites in cluster II. d The recruitment of RelA to different binding sites (BS) located in cluster II is tested by ChIP assay. The values are normalized to the input for each sample. e EMSA shows the combination between RelA and specific sequence. Binding complexes (arrow) are identified by supershift band with antibody against RelA. f Effects of binding-site mutagenesis on the RelA-mediated regulation. On the left side is a schematic representation of the normal (yellow) and site-directed altered (black) constructs. The right panel shows the luciferase activity normalized to Renilla luciferase activity. The construct with no site-directed alteration (WT) is used as control. Data are presented as mean ± standard deviation. **P < 0.01. ***P < 0.001; one-way ANOVA. Data are representative of three experiments done in triplicate

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