c-Myb regulates transcriptional activation of miR-143/145 in vascular smooth muscle cells
Fig 3
Chromatin immunoprecipitation (ChIP) of c-Myb on the miR-143/145 promoter of immortalized mouse carotid artery vascular smooth muscle cells (VSMC).
Cells were fixed, lysed and chromatin was fragmented into oligonucleosomes (200-1500bp) then immunoprecipitated overnight at 4°C with rabbit anti-c-Myb antibody (2mg, SC-517X, Santa Cruz), anti-histone H3 (2mg, Santa Cruz) or control rabbit serum (2mg, Santa Cruz). (A) The miR-143 and miR-145 promoter contains four c-Myb binding sites (MBS). (B) ChIP and PCR revealed specificity of c-Myb binding to MBS in carotid VSMC. As a negative control, ChIP was performed on a promoter situated ~15kb upstream of miR-143/145 with no known or predicted MBS. (C) qPCR reveals differential binding of c-Myb on predicted MBS of the miR-143/145 promoter. c-Myb preferentially binds MBS2 and MBS4 in carotid VSMC (p<0.01, one-way ANOVA). When normalized to histone H3 density, c-Myb preferentially binds MBS4 (p<0.001***, one-way ANOVA). ChIP was performed in n = 3 biological samples. qPCR analysis had three technical repeats for each biological sample.