Nuclear GSK3β more potently drives NF-κB activation than cytoplasmic GSK3β. (A) 293T cells were cotransfected with an NF-κB dependent Firefly luciferase construct, a Renilla luciferase vector, and either a vector control or the indicated GSK3β construct. Forty-eight hours posttransfection, cells were analyzed for luciferase signal. Data reported are fold change from the vector control signal and is normalized for Renilla signal (n = 3). (B) The indicated cells with or without targeted GSK3β induction were analyzed for NF-κB signaling components by western blot. Note the NLS isoform is at higher molecular weight because of the construct’s exogenous NLS. (C) HL60 and OCI-AML3 cells were fractionated into nuclear and cytoplasmic lysates and analyzed for p65 nuclear translocation by western blot. (D) Linear correlation analysis of nuclear localization scores of GSK3β and p65 in primary AML samples quantified using imaging cytometry (n = 82). (E) The indicated cells with or without targeted GSK3β induction were analyzed by western blotting for changes in the NF-κB-dependent survival genes, XIAP and Bcl-XL. ***P < .001.