NF-κB-Regulated miR-99a Modulates Endothelial Cell Inflammation

Figure 5

EMSA and ChIP verification of interactions of NF-κB with its binding sites in the miR-99a promoter. (a) EMSA validation of NF-κB binding sites on miR-99a promoter. Lane 1, labeled positive NF-κB probe only; lane 2, nuclear extracts plus labeled positive NF-κB probe; lane 3, labeled WT probe only; lane 4, nuclear extracts plus labeled WT probe; lanes 5 and 6, nuclear extracts plus labeled WT probe and cold unlabeled WT probe (50-fold and 100-fold labeled WT probe), respectively; lanes 7 and 8, nuclear extracts plus labeled WT probe and cold unlabeled Mut probe (50-fold and 100-fold labeled WT probe), respectively. NC: negative control; PC: positive control; NE: nuclear extracts; WT: wild-type probe; NWT: nonlabeled wild-type probe; NMut: nonlabeled mutant probe; P: free biotin-labeled probe. (b) Representative gel images of ChIP assay. (c) Relative NF-κB binding activity quantified by the ChIP bands. versus control group.

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