Challenges with Methods for Detecting and Studying the Transcription Factor Nuclear Factor Kappa B (NF-κB) in the Central Nervous System

Abstract

The transcription factor nuclear factor kappa B (NF-κB) is highly expressed in almost all types of cells. NF-κB is involved in many complex biological processes, in particular in immunity. The activation of the NF-κB signaling pathways is also associated with cancer, diabetes, neurological disorders and even memory. Hence, NF-κB is a central factor for understanding not only fundamental biological presence but also pathogenesis, and has been the subject of intense study in these contexts. Under healthy physiological conditions, the NF-κB pathway promotes synapse growth and synaptic plasticity in neurons, while in glia, NF-κB signaling can promote pro-inflammatory responses to injury. In addition, NF-κB promotes the maintenance and maturation of B cells regulating gene expression in a majority of diverse signaling pathways. Given this, the protein plays a predominant role in activating the mammalian immune system, where NF-κB-regulated gene expression targets processes of inflammation and host defense. Thus, an understanding of the methodological issues around its detection for localization, quantification, and mechanistic insights should have a broad interest across the molecular neuroscience community. In this review, we summarize the available methods for the proper detection and analysis of NF-κB among various brain tissues, cell types, and subcellular compartments, using both qualitative and quantitative methods. We also summarize the flexibility and performance of these experimental methods for the detection of the protein, accurate quantification in different samples, and the experimental challenges in this regard, as well as suggestions to overcome common challenges.

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“…Then, membranes were treated with enhanced chemiluminescence (ECL) utilizing the Bio-Rad Clarity Weston ECL blotting kit (Bio-Rad, Hercules, CA, USA), and visualized by the ChemiDoc. Relative quantification of OXPHOS protein levels was normalized to total protein as described previously [5,23].…”

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“…Salivary levels of the glucocorticoid cortisol have been found to be significantly higher upon awakening in night-shift workers [199], and a genome-wide study of differential methylation related to disruption of circadian rhythms during pregnancy, nightshift work has been found to result in hypermethylation of a CpG site in intron 3 (cg03709468; see iMethyl for location) in placental tissue of women who had engaged in such work [200]. This methylation site is within an NF-kB motif (Figure 7), which has been shown to be involved in responding to stress and to loss of sleep [201]. Methylation of this NF-kB site would be expected to block NF-kB binding to the motif [202].…”

“…Hypermethylation at a CpG site in intron 3 is found in placental tissue of pregnant women who worked on the nightshift[200]. The transcription factor NF-kB is predicted by the program Alibaba2 to bind at this site, and among other roles, it is involved in response to immune stimuli as well as stress from sleep deprivation[201,206], certainly consistent with nightshift work. Methylation of this NF-kB site would be expected to block NF-kB binding to the motif[202].…”

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“…It is known that the NF-κB pathway plays an important role in the modulation of inflammatory and immune responses. 25 Under normal conditions, NF-κB is found in the cytoplasm as the dimer complex p65/p50 with the kappa B inhibitor forming the NF-κB-IκB complex which involves TNF-α. As expected, the treatment with TNF-α induces IκB protein phosphorylation and the NF-κB dimer p65/p50 is translocated into the nucleus.…”

“…It is known that the NF-kB pathway plays an important role in the modulation of inammatory and immune responses. 25 Fig. 4 Cell cycle analysis with the control U87MG cells (above) and the treatment of 0.93 mM 4d (below).…”