I want to examine the effects of drugs by NF-kB activation.
I know that first signal is IkB phosphorylation by IKK and degradation, departing NF-kB (p65+p50).
And then NF-kB translocate to nucleus and activate to express genes.
I am trying to use the 4 methods to see NF-kB activation.
1. p65 expression in nucleus by immunocytochemistry (translocation)
2. p65 expression in nucleus by western blot
3. p-IkB expression in cytosol by western blot
4. IkB degradation in cytosol
But, some researchers showed p-p65 expression to see NF-kB expression.
Is it OK to see the p-p65 expression?
What is mechanism related to p-p65?
What is the best method to see NF-kB activation?
Thank you for reading :)
Dear Yoo-Lung,
Your suggestions are fine and complementary if you want to study the activation of NFkB. I would also suggest to test NFkB activity using a luciferase plasmid if possible and if your cells are easily transfected.
You don't need to use cytosolic extract to study the phosphorylation of IkBa or its degradation, it works well on whole cell extracts. Obviously, if you prepare cell fractions to show the nuclear translocation of p65 by WB, you might want to use the cytosolic fractions from the same experiment to run the WB for IkBa.
The phosphorylation of p65 is also driven by the IKK complex and has been shown to be essential for NFkB to have a full transcriptional activity on specific promoters. We have found that the phosphorylation on Ser536 is easier to detect than the phosphorylation on Ser276, but it might be cell type specific.
I would also suggest to use a positive control such as TNFa (1-100 ng/ml) as a positive control in your experiment, whatever the read-out. TNF should give you a phosphorylation of IkBa and p65 within 15 minutes, a clear degradation of IkBa within 30-45 minutes and a nuclear translocation of p65 within 1 hour.
All the best with your experiments,
Damien
Dear Yoo-Lung,
Your suggestions are fine and complementary if you want to study the activation of NFkB. I would also suggest to test NFkB activity using a luciferase plasmid if possible and if your cells are easily transfected.
You don't need to use cytosolic extract to study the phosphorylation of IkBa or its degradation, it works well on whole cell extracts. Obviously, if you prepare cell fractions to show the nuclear translocation of p65 by WB, you might want to use the cytosolic fractions from the same experiment to run the WB for IkBa.
The phosphorylation of p65 is also driven by the IKK complex and has been shown to be essential for NFkB to have a full transcriptional activity on specific promoters. We have found that the phosphorylation on Ser536 is easier to detect than the phosphorylation on Ser276, but it might be cell type specific.
I would also suggest to use a positive control such as TNFa (1-100 ng/ml) as a positive control in your experiment, whatever the read-out. TNF should give you a phosphorylation of IkBa and p65 within 15 minutes, a clear degradation of IkBa within 30-45 minutes and a nuclear translocation of p65 within 1 hour.
All the best with your experiments,
Damien
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