miR-181a decreases NF-κB binding to DNA-κB motif. (A) Western blot of cytoplasmic (Cyto.) or nuclear (Nuc.) fractions. The protein bands (NIH software; ImageJ) were normalized to β-actin or SP1, cytoplasmic and nuclear fraction markers, respectively (left). Graphical representations in U2932 cells (3 independent experiments) (right). Statistical significance was determined by Student t test and the results are presented as mean ± SD. (B) Quantitative ELISA-based assay of NF-κB transcription factors binding activity. Nuclear extracts (10 μg) were prepared as in panel A from the control or miR-181a inducible cell lines and incubated with the noted NF-κB antibodies. Standard curve was prepared with recombinant NF-κB P50 protein. WT consensus oligos or MUT oligos were used as positive and negative controls, respectively. Shown are cumulative results from 3 independent experiments; mean ± SEM. (C) EMSA as previously described.²⁴  Briefly, double-stranded NF-κB consensus oligos were end-labeled with [³²P]-adenosine triphosphate and incubated with U2932 nuclear extracts. For the super shift analyses, extracts were incubated with anti-P50 or anti-CREL antibodies. The DNA-protein complexes were resolved by electrophoresis, the gels were dried, and analyzed by autoradiography. The arrows indicate the super shift bands. See also insert (top right) for lanes 5 and 6 CREL super shift. Densitometry-based analysis of protein bands normalized to the loading control Oct-1 is shown. (D) Quantitative real-time–PCR analyses of NF-κB target genes in HBL1 cells transfected with GFP control or GFP–miR-181a plasmids (24 hours), and GFP sorted by FACS. Values are presented as mean ± SEM (n = 3; 2 independent experiments). Shown are P50/CREL transcription complex specific NF-κB target genes that either contain (i) or lack (ii) putative miR-181a binding sites (Probability of Interaction by Target Accessibility algorithms). (E) Ratios of NF-κB P50 activity (as in [B] above) and quantitative real-time–PCR levels of miR-181a expression in primary GCB and non-GCB tumor samples. (F) Schematic diagram of study results showing the effect of differential miR-181a expression (low [i] and high [ii]) on the regulation of NF-κB signaling pathway. Red shadings indicate experimentally validated targets of miR-181a. In panels A and B, the Student t test statistical significance is as follows: *P < .05; **P < .01; ***P < .001; ****P < .0001. WT, wild-type.