Figure 5

miR-133a directly down-regulates GRHL2 expression. (a) The 3′UTR of GRHL2 contains a putative miR-133a binding site. (b) Beas-2b cells were mock transfected or transfected with control or miR-133a mimics and cultured for 3 days. Cells were harvested for western blot analysis of GRHL2 protein and GAPDH was used as an internal control. Experiments were conducted at least three times, and a representative result is shown. The blots in (b) were cropped from different parts of the same gel. Unprocessed original scans of the blots are shown in Supplementary Fig. S5. (c) The 3′UTR of GRHL2 and its mutant (3′UTR-133a/M) were cloned into the pmirGLO vector and then transfected with control or miR-133a mimics into HEK293 cells. (d) Cells were harvested for firefly luciferase (fLuc) assays with Renilla luciferase (rLuc) as internal control. The relative luciferase activity in cells transfected with pmirGLO control vector and control mimics were normalized to 1. Data are mean ± S.E. (n = 3) with **P < 0.01.