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. Author manuscript; available in PMC: 2010 Jun 1.
Published in final edited form as: Arterioscler Thromb Vasc Biol. 2009 Apr 2;29(6):921–928. doi: 10.1161/ATVBAHA.109.187229

Figure 4. DN-Brg1 blocks the ability of myocardin to increase SRF binding to the promoters of smooth muscle-specific genes within chromatin.

Figure 4

A–B. B22 cells (an NIH3T3 cells line that inducibly express DN-Brg1 in response to tetracycline withdrawal13) grown in the presence (control) or absence (induced DN-Brg1) of tetracycline were transduced with myocardin or YFP control adenovirus. After 30 hrs, cells were fixed and harvested for chromatin immunoprecipitation assays. Chromatin was precipitated using an antibody against SRF or using IgG negative control. The precipitated genomic DNA was purified and the presence of the promoters of SRF-dependent genes measured by real time PCR using gene specific primers8. A. The increase in SRF binding in samples transduced with myocardin is indicated relative to those transduced with YFP. These data were calculated and normalized to input levels as follows: Relative SRF binding=2−ΔΔCt, with ΔΔCt= (Ct myocardin-Ct input)-(CtYFP-Ct input). B. The relative inhibition of myocardin induced SRF binding by DN-Brg1 is shown. This was calculated as follows: Relative SRF binding= 2−ΔΔCt, with ΔΔCt= (Ct DN-Brg1+Myocardin -Ct input)-(Ct YFP+Myocardin -Ct input). Data shown in panels ‘A’ and ‘B’ are the mean±SEM of 7 samples obtained from 3 independent experiments. A one-way t-test was performed and the asterisks indicate the results that are statistically different from 1 (P<0.05). C–D. Primary colon smooth muscle cells were prepared from 4 week old mice. The cells were transduced by DN-Brg1 or YFP control adenovirus. After 36 hours, cells were fixed and harvested for chromatin immunoprecipitation assays as above. C. The relative Brg1 binding to several SRF dependent genes in control primary smooth muscle cells is shown. The Brg1 binding to promoters were normalized to IgG control. The relative Brg1 binding was calculated as RQ=2−ΔCt, with ΔCt= Ct Brg1-Ct IgG. D. The relative inhibition of SRF binding by DN-Brg1 is shown. The inhibition of SRF binding by DN-Brg1 is calculated as, RQ=2−ΔΔCt, with ΔΔCt= (Ct DN-Brg1-Ct input)-(Ct YFP-Ct input). Data presented are the mean ±SEM of 4 samples.