Systematic detection of noncanonical NF-κB activation.
In unstimulated cells, NF-κB dimers usually exist as latent complexes in the cytoplasm with the IκB (inhibitor of NF-κB) proteins or IκB-like protein p100, the precursor of NF-κB2 mature form p52. Accordingly, there are two major mechanisms leading to NF-κB activation: inducible degradation of IκBs and processing of p100 to generate p52 (selective degradation of the C-terminal IκB-like sequence of p100), which are termed the canonical and noncanonical NF-κB pathways, respectively. While activation of the canonical NF-κB pathway plays critical roles in a wide range of biological processes, the noncanonical NF-κB pathway has important but more restricted roles in both normal and pathological processes. Systematic detection of the noncanonical NF-κB pathway activation is very important for understanding the physiological role of this pathway in biological processes, and for the diagnosis, prevention, and treatment of related diseases. We describe here the methods we employ to detect noncanonical NF-κB activation in cells and tissues. These methods are immunoblotting, co-immunoprecipitation, immunofluorescence, immunohistochemistry, chromatin immunoprecipitation (ChIP) analysis, and electrophoretic mobility shift assay (EMSA). Noncanonical NF-κB-induced gene expression changes can be determined by gene array analysis and quantitative real-time PCR.
Department of Microbiology and Molecular Genetics, University of Pittsburgh, School of Medicine, 1.18 Hillman Cancer Center, 5117 Centre Avenue, Pittsburgh, PA, 15213, USA.
Zhaoxia
Z
Qu
Gutian
G
Xiao
D015153:Blotting, Western / Q000379:methods; D047369:Chromatin Immunoprecipitation / Q000379:methods; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D047468:Immunoprecipitation / Q000379:methods; D066298:In Vitro Techniques; D016328:NF-kappa B / Q000378:metabolism*; D052003:NF-kappa B p52 Subunit / Q000378:metabolism; D020411:Oligonucleotide Array Sequence Analysis; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction
D016328:NF-kappa B; D052003:NF-kappa B p52 Subunit