Figure 4

Inhibition of MYB and MYBL1 protein expression by p95HER2 and miR-503. (A) MYB family protein expression in vector- and p95HER2-overexpressing cells. Cells were lysed 48 h after induction and subjected to Western blotting. β-actin is shown as loading control. The graph shows mean values with S.E.M. error bars of 5 independent, parallel experiments, normalized to the β-actin loading control. *Significantly different from the corresponding value in vector cells, p < 0.05. (B) Effect of miR-503 mimics on MYB family protein expression in vector cells. Immediately after induction, cells were transfected with miR-503 or negative control miRNA mimics. 48 h later, cells were lysed and subjected to Western blot analysis of MYB, MYBL1 and MYBL2 expression. β-actin is shown as a loading control. The graph shows mean values with S.E.M. error bars of 3 independent, parallel experiments, normalized to the β-actin loading control. *Significantly different from the corresponding value in vector cells, p < 0.05. (C) Effect of miR-503 mimics on MYBL1 3′UTR regulation of upstream renilla luciferase activity. The psiCHECK2 plasmid containing the wild-type 3′UTR of MYBL1 was cotransfected into vector and p95HER2 cells with 40 nM of miR-503 or negative control miRNA mimics. 48 h after transfection, cells were lysed and luciferase activity measured as described in Materials and Methods. Data was normalized to firefly luciferase activity and are shown as the log2 transformed change relative to empty psiCHECK2 vector. The graph shows mean values with S.E.M. error bars of 11–12 independent, parallel experiments. * and **Significantly different from corresponding negative control miRNA values, p < 0.05 and 0.01, respectively.