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. Author manuscript; available in PMC: 2017 Jan 19.
Published in final edited form as: Immunol Cell Biol. 2016 Jul 19;95(1):42–55. doi: 10.1038/icb.2016.63

Figure 3. Electrophoretic mobility shift assay of Stat3 and C/EBPβ binding specificity to the miR-21 and miR-181b promoters.

Figure 3

EMSA was performed to determine the Stat3 and C/EBPβ binding specificity to the miR-21 and miR-181b promoters. Nuclear proteins were isolated from CD31+ cells purified from the bone marrow Gr1+CD11b+ cells from late septic mice. Protein extract was incubated with biotin-labeled double-stranded DNA oligos containing the Stat3 or C/EBP binding sites in the miR-21 and miR-181b promoters. The protein-DNA complexes were then electrophoresed on 6% native polyacrylamide gel, immunoblotted, and the DNA was visualized with streptavidin-conjugated antibody and chemiluminescent reagent. (A) Stat3 and C/EBPβ binding to miR-21 promoter. In lane 3, 3 μg of antibody specific to p-Stat3 or C/EBPβ was incubated with the nuclear protein for 5 min before the addition of the labeled DNA probe. In lanes 5 and 6, 200-fold excess of unlabeled wild-type or mutated probe, respectively, was used as a competitor to confirm the binding specificity. Results are representative of three experiments. (B) Stat3 and C/EBPβ binding to miR-181b promoter. The short arrow with an asterisk indicates a non-specific band, which migrated at the same rate as the supershifted complex. The results are representative of two experiments.