Hello Mohsen,

Alongwith determining the expression of NF-kB targets by real-time PCR

You can evaluate NF-kB activation by Western blot analysis for:

Degradation of cytosolic IkBalpha

Level of cytosolic pIKK

Nuclear accumulation of NF-kBp65

Ikba is indeed an NFkB target transcript (eg. In response to LPS/IL1) and can be used as an indicator of NFkB activation. However some additional proof as suggested by Nupur will be good to have.

Hi Mohsen,

take a look at Trans-AM NF-B assay (active motif), in my opinion it works really well. Through this assay, you should detect nuclear translocation of different complexes of NF-kB in order to be sure of its activation, as suggested by Nupur as well.

Depending on your project you can evaluate NF-kB p65 activity in many ways:

Using Western Blot:

- Phospho-NF-kB p65 (Ser 536) and total NF-kB p65 levels

- Measure phospho IkBalpha and total IkBalpha levels (If canonical NF-kB pathway is activated, it would be degraded)

-Phospho IKKb and total IKKb levels (Caution, I have used both Cell Signalling and Abcam antibodies with little success)

- You can also look at nuclear localization of NF-kB (Under IF and/or IHC)

-You can also quantify expression of adhesion molecules (ICAM-1, VCAM-1, and e-selectin), cytokines (IL-1beta, IL-6, MIP1alpha, IP-10) using quantitative real time PCR and/or Western Blot. You should also include NF-kB inhibitors such as TPCA-1 and JSH-23

You should also include proper controls for NF-kB p65 such as LPS or TNFa