miRNA-15a and -16 target NF-κB in MM cells. (A) MM cells (pre-miRNA-15a–, -16-1 probe–, control probe–transfected MM.1S) were harvested at 24 hours after transfection and treated with and without TNF-α (10 ng/mL) for 20 minutes. Whole cell lysates were subjected to Western blotting using anti-TAB3, phospho(p)-TAK1, and -actin antibodies; nontransfected MM.1S cells were used as control (ctrl). (B) MM cells (pre-miRNA-15a–, -16-1 probe–, control probe–transfected MM.1S) were harvested at 24 hours after transfection and treated with and without TNF-α (10 ng/mL) for 20 minutes; nontransfected MM.1S cells were used as control (ctrl). NF-κB-p65, p50, -p52, -RelB transcription factor-binding to its consensus sequence on the plate-bound oligonucleotide was measured in nuclear extracts. All results represent means (± SD) of triplicate experiments. (C) MM cells (pre-miRNA-15a–, -16-1 probe–, control probe–transfected MM.1S) were harvested at 12 hours after transfection and treated with and without TNF-α (10 ng/mL) for 20 minutes. Nuclear and cytoplasmic protein extracts were subjected to Western blotting using anti–phospho(p)-65, -p50, -p52, -RelB, -nucleolin, –p-IkB, and -actin antibodies; nontransfected MM.1S cells were used as control (ctrl). (D) MM cells (pre-miRNA-15a–, -16-1 probe–, control probe–transfected MM.1S) were harvested at 24 hours after transfection and treated with and without TNF-α (10 ng/mL) for 20 minutes; nontransfected MM.1S cells were used as control (ctrl). Immunocytochemical analysis was assessed using anti–phospho-NF-κB-p65 antibody, with DAPI used to stain nuclei.