miR-181a directly targets several NF-κB signaling components. (A-B) Representative western blot analyses in DLBCL cell lines transfected with 2 μg precursor control (−), miR-181a (+), or miR-181a and anti–miR-181a by electroporation, and analyzed after 24 hours. Densitometry analyses of the protein bands were normalized to the loading control β-actin (NIH software; ImageJ). The relative band density values in (+) or anti-miR samples were compared with the controls (set as 1) and are noted below each blot. (A, right panel) Quantitative real-time–PCR miR-181a assay indicating the fold change in miR-181a expression between the control and miR-181a transfected samples as an assessment of transfection efficacy (2 independent experiments in triplicates). (C) Quantitative real-time–PCR analyses of NF-κB gene expression in cells transfected with control or miR-181a green fluorescent protein (GFP) plasmids for 24 hours. To enrich for transfected cells, the GFP+ cells were sorted and RNA prepared. Shown are the relative changes in gene expression from 2 independent experiments in triplicates. Values are presented as mean ± standard deviation (SD). Statistical significance was determined by Student t test. *P < .05. (D) Dual luciferase reporter assay in HeLa cells co-transfected with plasmids containing the noted WT or MUT 3′UTR sequence harboring putative miR-181a binding sites, and control or miR-181a plasmids. The firefly signals were normalized with the internal control renilla luciferase. Shown are the firefly/renilla ratios for each 3′UTR construct. The experiments were repeated 3 to 6 times in triplicates (mean ± SEM), and statistical significance analyzed by Student t test. ****P < .0001. ns, not significant; WT, wild-type.