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

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

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

IKKα and IKKβ knock-down in Hela cells leads to diminished IκB degradation and p65 phosphorylation.

<p>HeLa cells were grown in 6-well plates and transfected with the indicated siRNA for 3 days. Western blots were performed on total cell extracts after treatment with TNF for the indicated times. Tubulin levels are shown as a loading control.</p

IKKβ kinase mutant inhibits TNF and IKKα-induced NF-κB-dependent reporter gene activity.

<p>WT and IKKβ null MEFS were transfected with the indicated vector construct and with the NF-κB-dependent luciferase reporter. Luciferase luciferase activity was measured 48 hr after transfected. Where indicated, cells were treated with TNF for 4 hrs. Relative luciferase values were calculated using a renilla control expression vector for normalization. Relative luciferase values are normalized to vector control samples.</p