Assaying Homodimers of NF-κB in Live Single Cells

• Autores: Martin, Erik W.; Chakraborty, Sayantan; Presman, Diego Martin; Tomassoni Ardori, Francesco; Oh, Kyu Seon; Kaileh, Mary; Tessarollo, Lino; Sung, Myong Hee
• Año de publicación: 2019
• Idioma: inglés
• Tipo de recurso: artículo
• Materia: DIMERIZATION; MICROSCOPY; NF-ΚB; NUMBER AND BRIGHTNESS; OLIGOMERIZATION; RELA; TRANSCRIPTION FACTOR
• Institución: Consejo Nacional de Investigaciones Científicas y Técnicas

NF-κB is a family of heterodimers and homodimers which are generated from subunits encoded by five genes. The predominant classical dimer RelA:p50 is presumed to operate as “NF-κB” in many contexts. However, there are several other dimer species which exist and may even be more functionally relevant in specific cell types. Accurate characterization of stimulus-specific and tissue-specific dimer repertoires is fundamentally important for understanding the downstream gene regulation by NF-κB proteins. In vitro assays such as immunoprecipitation have been widely used to analyze subunit composition, but these methods do not provide information about dimerization status within the natural intracellular environment of intact live cells. Here we apply a live single cell microscopy technique termed Number and Brightness to examine dimers translocating to the nucleus in fibroblasts after pro-inflammatory stimulation. This quantitative assay suggests that RelA:RelA homodimers are more prevalent than might be expected. We also found that the relative proportion of RelA:RelA homodimers can be perturbed by small molecule inhibitors known to disrupt the NF-κB pathway. Our findings show that Number and Brightness is a useful method for investigating NF-κB dimer species in live cells. This approach may help identify the relevant targets in pathophysiological contexts where the dimer specificity of NF-κB intervention is desired.
Fil: Martin, Erik W.. National Institutes of Health; Estados Unidos
Fil: Chakraborty, Sayantan. National Institutes of Health; Estados Unidos
Fil: Presman, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Fisiología, Biología Molecular y Neurociencias. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Fisiología, Biología Molecular y Neurociencias; Argentina
Fil: Tomassoni Ardori, Francesco. National Institutes of Health; Estados Unidos
Fil: Oh, Kyu Seon. National Institutes of Health; Estados Unidos
Fil: Kaileh, Mary. National Institutes of Health; Estados Unidos
Fil: Tessarollo, Lino. National Institutes of Health; Estados Unidos
Fil: Sung, Myong Hee. National Institutes of Health; Estados Unidos