Down-regulation of IKKα triggers significant growth inhibition without altering NF-κB activity. (A) MM.1S and OPM1 cells were transfected with either scrambled (Sc) or 2 different IKKα shRNA. Whole-cell lysates were immunoblotted with anti-IKKα, IKKβ, and GAPDH Abs. (B) Nontransfected (non, ●), scramble-shRNA transfected (Sc, ■), IKKα sh1 (▴), or IKKα sh2 (♦) transfected MM.1S (left panel) and OPM1 (right panel) cells (0.25 × 106/mL) were cultured for 48 hours. Cell viability was determined by trypan blue exclusion. (C) MM.1S and OPM1 cells were transfected with Sc or IKKα shRNAs. Nuclear extracts were subjected to EMSA to assess NF-κB activity. The intensity of the bands was digitalized by ImageJ software and indicated as fold increase relative to nontransfected cells. (D) To determine the role of Rel family proteins mediating NF-κB activity in OPM1 cells before and after IKKα shRNA 1, supershift assays were carried out using anti-p65, p50, p52, and RelB (R-B) Abs. (E) MM.1S cells were transfected with Sc or IKKα shRNAs. Whole-cell lysates were immunoblotted with anti-p-IKKα/β, -β-catenin, -Aurora-A, and -GAPDH Abs. (F) MM.1S cell lysates were immunoprecipitated with anti-GFP, -IKKα, or -β-catenin Abs and then immunoblotted with anti-IKKα or -β-catenin Abs. Whole-cell lysates (W.L.) served as a protein loading control for IKKα and β-catenin.
