(A) WT clone (A3) and two independent IKKα KO clones or (B) WT clone (A3) and two independent IKKβ KO clones were seeded in their normal growth medium for 48 h, prior to treatment with 10 ng/ml recombinant TNFα for the indicated timepoints. Whole-cell extracts were prepared, fractionated by SDS–PAGE and Western blotted with the indicated antibodies. Similar results were seen for the two other WT clones (A8 and E10) and the other IKKα (A2) and IKKβ (A4) KO clones (data not shown). (C and D) WT, IKKα KO, IKKβ KO and IKKα/β DKO HCT116 cells were seeded in antibiotic-free growth medium overnight prior to transient transfection with NF-κB-RE firefly luciferase and renilla luciferase plasmids. The following day, cells were treated with 10 ng/ml recombinant TNFα for the indicated time periods. Firefly luciferase was normalised to renilla luciferase and data expressed as log₂(fold change in TNFα-induced luciferase activity relative to the relevant, matched untreated condition). Results are mean ± SD of three independent experiments, each of which was performed with three independent clones, seeded in triplicate (WT — A3, A8 and E10. IKKα KO — F6, C7, A2. IKKβ KO — G9, A7, A4 and IKKα/β DKO — C8, G1, E9). Significance testing was performed using two-way ANOVA (repeated measures) with Tukey post-hoc test. P values relate to comparison with corresponding timepoint(s) of WT control. P < 0.0001 (***).