IKKα and IKKβ each contribute to TNF-induced NF-κB activity in HeLa cells.

<p>HeLa cells, seeded in 24-well plates were transiently transfected with indicated siRNA constructs for 48 hr. Then media was replaced and cells were further transfected with NF-κB responsive 3x-κB luciferase and a control Renilla luciferase contructs. TNF was added (as indicated) and 24 hr later cells were lysed and dual lucifearse assay was performed. Luciferase readings in untreated and control vector transfected cells were normalized as 1.</p

NF-κB DNA binding activity is reduced in IKKα and in IKKβ deficient cells.

<p>DNA binding activity of NF-κB was measured by gel shift assay. Indicated MEF cells were treated with TNFα for indicated times. Nuclear proteins were subject to gel shift assay for DNA binding analysis.</p

The role of IKKα and IKKβ in p65 phosphorylation and IκBα degradation in response to TNFα.

<p>MEF cells that are deficient for IKKα, IKKβ, or both IKKα and IKKβ (DKO) were treated with TNFα for the indicated times. NF-κB activity, as measured by IκBα degradation and p65 phosphorylation, is diminished in IKKα and IKKβ deficient MEF cells. IKKα and IKKβ DKO cells show no detectable p65 phosphorylation. Tubulin levels are shown as a loading control.</p