Hello,
I understand the general mechanism of how NFkB gets liberated from its IKK to enter the nucleus.
But, does NFkB itself in T cells get phosphorylated when it was still in cytoplasm, once inside the nucleus, or both?
By the same token, what are the most effective assays (flow/western blot/etc.) to determine the level of nuclear and cytoplasmic NFkB expression in CD4 T cells?
Thank you in advance!
Hi,
In the latest publications about NFkB signalling, there are no mentions about any phosphorylation in the nucleus.
To analyse differential expression of NFkB in T-cells, you can perform a differential centrifugation to isolate nuclei, then extract your protein content from the nuclei and separate it on a western-blot = antibodies anti-NFkB P65 are available.
To determine the quantity of NFkB in the cytoplasm, you realize the same experiment on whole cells, and you substract the quantity of NFkB in the nucleus (obviously, on the same population of T-cells).
Hope it helps,
Sincerely.
The dogma is that NFkb gets phosphorylated in the cytoplasm and get trafficked to the nucleus. But seems there are exceptions.
Paper on NFkB signaling: Article The Nuclear Signaling of NF-κB – Current Knowledge, New Insi...
Here is a paper which mentions nuclear phosphorylation of NFkB (Fig. 5):
Article Ser276 phosphorylation of NF-kB p65 by MSK1 controls SCF exp...
Most effective techniques to determine nuclear vs cytoplasmic phosphorylation:
1. Western Blot: Use a cell fractionation kit to get nuclear/cytoplasmic lysates. Run western blot on them using antibodies directed against total/phosphorylated NFkB.
http://www.jimmunol.org/content/jimmunol/188/3/1213.full.pdf
2. Microscopy: Fluorescence/Confocal microscopy using antibodies directed against phosphorylated NFkB and nucelar/cytoplasmic stains.
Article Quantitative imaging assay for NF-κB nuclear translocation i...
3. Imaging Flow Cytometry: Regular Flow is not sensitive enough to show you nuclear vs cytoplasmic expression of proteins. This is where ImageStream comes in. See paper below for details.
Article Simultaneous assessment of NF-κB/p65 phosphorylation and nuc...
Below is a paper comparing all 3 methods:
Article Quantifying Nuclear p65 as a Parameter for NF-kappa B Activa...
Hello Quang.
Immunocytochemistry, followed by Western blot of cytoplasmic and nuclear fractions simultaneously. The cells should come from a single experiment and be divided into two portions for each analysis.
Yes, both cytoplasmic and nuclear p-NF-kB are found with our studies of p-p65NF-kB[Thr] antibody. p-p65NF-kB[Ser] antibodies are also found in both fractions, depending on the position of Ser residues.
Best wishes.
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