Hi,
I have lysate from human macrophages, and I wonder what will be the best way to check the NFKB activation in those cells.
I was thinking p-p65 or p-ikkb./
Is that make sense?
Dear Dr. Shani Dror
Yes, you are right. It does make sense.
In unstimulated cells, NF-κB subunits are restricted to the cytoplasm due to the inhibitory effects of the inhibitor of κB (IκB) family. IκBα or IκBβ selectively binds to the p50/p65 heterodimer and masks their nuclear localization signal (NLS), preventing their nuclear translocation.
Stressful stimuli induce NF-κB signaling through several cell membrane-bound receptors that converge on the activation of the IκB kinase (IKK) complex. The activated IKK complex phosphorylates IκBα, priming it for subsequent ubiquitylation and proteasome-mediated degradation. Disassociation of IκBα induces NF-κB heterodimers to locate in the nucleus and bind to specific gene promoters to modulate the expression of pro- and anti-inflammatory proteins. As a negative feedback loop, the expression of IκBα and IκBβ is up-regulated by the NF-κB heterodimers, to terminate the signaling pathway.
For Western Blot analysis, you may perform a time-dependent experiment, namely, at 0, 10, 20 and 30 mins for the NF-κB/p65 translocation following activation. You may find that phosphorylated p65 may occur initially in the cytosol, with only a minor fraction which may/may not be found in the nucleus. At later time points, after stimulation, you may observe phosphorylated p65 in the nucleus and may detect none in the cytosol. You should also look out for phosphorylated IκBα in the cell lysate. You may observe phosphorylated IκBα (pIκBα) at the initial time points.
Please note that you need to add protease inhibitors (to prevent protein degradation) and phosphatase inhibitors (to prevent dephosphorylation), fresh, just before cell lysis. This is because in an intact cell, protease and phosphatase enzymes are typically sequestered in cellular compartments. However, during the process of cell lysis, these compartments are broken open, releasing proteases and phosphatases that may degrade proteins and cause changes in phosphorylation states.
I have attached a few references which may help!
Article Monitoring the Levels of Cellular NF-κB Activation States
Article Quantifying Nuclear p65 as a Parameter for NF-kappa B Activa...
Article Phosphorylation of RelA/p65 on Serine 536 Defines an I B -in...
Regards,
Malcolm Nobre
I am not an expert in this field, but I am very interested and have researched to find an answer. I received some assistance from tlooto.com for this response. Could you please review the response below to see if it is correct?
To test NF-κB activation via Western blot in human macrophage lysates, detecting phosphorylated p65 (p-p65) is a suitable choice, as p65 phosphorylation is a key indicator of NF-κB activation [3][5]. Additionally, phosphorylated IκB kinase β (p-IKKβ) can also be assessed, as it plays a critical role in the activation pathway by phosphorylating IκBα, leading to its degradation and subsequent release of NF-κB [1][2]. Both markers provide insights into different stages of the activation process. Ensure the use of specific antibodies against p-p65 and p-IKKβ, and include appropriate controls for accurate interpretation of results [4][5].
Reference
[1]Martínez, F., Helming, L., & Gordon, S. (2009). Alternative activation of macrophages: an immunologic functional perspective.. Annual review of immunology, 27, 451-83 .
[2]Snodgrass, R., Boss, M., Zezina, E., Weigert, A., Dehne, N., Fleming, I., Brüne, B., & Namgaladze, D. (2015). Hypoxia Potentiates Palmitate-induced Pro-inflammatory Activation of Primary Human Macrophages*. The Journal of Biological Chemistry, 291, 413 - 424.
[3]Jiang, Y., Huang, K., Lin, X., Chen, Q., Lin, S., Feng, X., Zhen, C., Huang, M., & Wang, S. (2017). Berberine Attenuates NLRP3 Inflammasome Activation in Macrophages to Reduce the Secretion of Interleukin-1β.. Annals of clinical and laboratory science, 47 6, 720-728 .
[4]Ghiboub, M., Zhao, J., Yim, A. L. L., Schilderink, R., Verseijden, C., Hamersveld, P. V. v., Duarte, J., Hakvoort, T., Admiraal, I., Harker, N., Tough, D., Henneman, P., Winther, M. D. d., & Jonge, W. D. d. (2020). HDAC3 Mediates the Inflammatory Response and LPS Tolerance in Human Monocytes and Macrophages. Frontiers in Immunology, 11.
[5]Fontaine, C., Rigamonti, E., Nohara, A., Gervois, P., Teissier, E., Fruchart, J., Staels, B., & Chinetti-Gbaguidi, G. (2007). Liver X Receptor Activation Potentiates the Lipopolysaccharide Response in Human Macrophages. Circulation Research, 101, 40-49.
Malcolm Nobre Thank you Malcolm for your very informative and helpful answer.
Just to clarify - for the p-65 do I need to separate the cytosol from the nucleus? otherwise, it will be hard to get a clear answer.
Is it the same for the ikkb?
Thanks a lot!
Dr. Dror,
Yes, you will have to separate cytosol from the nucleus for p65 but not for ikkb.
Regards.
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