miR-181a represses the NF-κB signaling activity. (A) Dual luciferase NF-κB reporter activity assay. DLBCL cell lines were co-transfected with the NF-κB luciferase reporter plasmid κB-TATA, renilla vector, and miR-181a, or control plasmids (24 hours). Three independent experiments in triplicates were analyzed per cell line. A nontargeting miRNA control (miR-29c) transfection in VAL cells is noted in gray. Results are presented as mean ± SEM. Statistical significance was determined by Student t test. (B) Dual luciferase NF-κB reporter activity assay in DLBCL cell lines. Cells were transfected with control or miR-181a. Twenty-four hours post-transfection, cells were counted and transfected again with either control or anti–miR-181a oligos (Ambion). At the 48-hour time point, the luciferase signal was analyzed. Three independent experiments in triplicates per cell line. (C) Anti-IKKγ immunoprecipitation. HEK-2932 or HBL1 cells were transfected with either miR-181a or control (24 hours), and immunoprecipitated from cellular protein extract with anti-IKKγ antibody or control IgG. Shown are the immunoblots with anti-ubiquitin antibody. The noted arrows indicate the poly ubiquitinated IKKγ bands (>50 kilodalton [kDa]). The IKKγ input controls are shown below. On the far right, a graphic representation of the relative ubiquitin bands density normalized to the IKKγ input from 3 independent experiments in the HEK-293 cells is shown. Statistical significance was determined by Student t test. Results are presented as mean ± SD. *P < .05; ***P < .001; ****P < .0001. ns, not significant.