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. 2008 Dec 31;28(53):14341–14346. doi: 10.1523/JNEUROSCI.2390-08.2008

Figure 2.

Figure 2.

miR-9 targets the 3′ UTR of REST. A, Cotransfection of HEK 293 cells with luciferase reporters containing the 3′ UTR of REST in the presence of increasing concentrations (0.1, 1.0, 5.0, or 15.0 nm) of artificial pre-miR-9 reduces relative luciferase activity (n = 4). B, C, Knockdown of REST following transfection of HEK 293 cells with pre-miR-9 (30 nm). A representative Western blot (B) and the densitometry from (n = 4) independent experiments (C) after normalization to β-actin. Data are mean ±SEM; *p < 0.05, **p < 0.01. D, A representative Western blot showing that target site protectors (TSP) designed to block the predicted miR-9 binding site of REST (TSP +) rescues mU6 miR-9/9*-mediated knockdown of REST. HEK 293 cells were cotransfected with mU6 miR-9/9* and TSP+ or TSP negative control (Neg) and REST levels measured in cell lysates 24 h later. Densitometry from n = 4 independent experiments indicate increased REST in TSP+ (0.97 ± 0.065)-treated versus TSP Neg (0.54 ± 0.212)-treated cells.