Selective IKKβ inhibition confirms a prominent role for IKKα in canonical NF-κB signalling.
(A) WT, IKKα KO and IKKβ KO HCT116 cells were seeded for 48 h prior to treatment with 10 µM BIX02514 or DMSO vehicle control for 30 min. Cells were then treated with TNFα (10 min) and processed for western blot. (B) WT, IKKα KO and IKKβ KO HCT116 cells were transiently transfected with reporter plasmids. The following day, cells were treated with BIX02514 or DMSO vehicle control for 30 min, prior to treatment with TNFα for 2 or 4 h. Firefly luciferase luminescence was normalised as above and expressed as log2(fold change in TNFα-induced luciferase activity relative to the relevant, matched untreated condition). Results are mean ± SD of two independent experiments using the same KO clones as in (A) each performed in technical triplicate. Significance testing was performed using two-way ANOVA (repeated measures) with Tukey post-hoc test. P < 0.0001 (***). BIX, BIX02514.