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. 2019 Dec 20;7:351. doi: 10.3389/fcell.2019.00351

FIGURE 4.

FIGURE 4

EMSA and ChIP-qPCR assay of the physical binding of Smad4 to the promoter regions of miR-375, miR-26a, and Ngn3. (A,D,G) Diagrams indicating the predicted binding sites for Smad4 within the promoters of miR-375, miR-26a, and Ngn3. (B,E,H) EMSA of the physical binding of Smad4 to the promoter regions of miR-375, miR-26a, and Ngn3. The promoter Smad4-binding sites with the highest scores based on bioinformatic predictions were selected for EMSA probe design. Following induction, cell nuclear extracts were prepared and incubated with the specific biotinylated EMSA probe for Smad4. Non-biotinylated EMSA probes were used as competitor probes. Smad4 was observed to bind to each of the three predicted binding sites incorporated into the EMSA probes. Furthermore, incubation with specific antibodies against Smad4 clearly resulted in supershifted bands. Probes containing mutated binding sites (mS4BS) were unable to compete for binding. S4BS, Smad4-binding site. (C,F,I) The amounts of Smad4 enriched within the promoter regions of miR-375, miR-26a, and Ngn3 in uninduced and induced MSCs were quantified by ChIP-coupled real-time qPCR. The percentages of input were calculated according to the threshold cycle values (CT). Non-specific IgG was used as the negative control. The amounts of immunoprecipitated chromatin were significantly higher for the induced MSCs than the uninduced MSCs, with a fold change in occupancy of 8.36, 7.89, and 6.61 for miR-375, miR-26a, and Ngn3, respectively. Values represent the mean ± SEM, n = 3.