NFκB Pathway

About NFkB Pathway:

NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) is a protein complex that controls transcription of DNA, cytokine production and cell survival. NF-κB plays a key role in inflammation, immune response, cell proliferation and protection against apoptosis, involves in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet irradiation, oxidized LDL, and bacterial or viral antigens. Incorrect regulation of NF-κB has been linked to cancer, inflammatory and autoimmune diseases, septic shock, viral infection, and improper immune development. NF-κB has also been implicated in processes of synaptic plasticity and memory.

There are five proteins in NFkB family including RelA, RelB, Rel, NF-κB1 (p105 / p50) and NF-κB2 (p100 / p52). A variety of extracellular signals can activate the enzyme IκB kinase (IKK). IKK, in turn, phosphorylates the IκBα protein, which results in ubiquitination, dissociation of IκBα from NF-κB, and eventual degradation of IκBα by the proteosome. The activated NF-κB is then translocated into the nucleus where it binds to specific sequences of DNA called transcriptional response element (NFkB-TRE). The DNA/NF-κB complex then recruits other proteins such as coactivators and RNA polymerase, which transcribe downstream DNA into mRNA, which, in turn, is translated into protein, which results in a change of cell function.

Detect NFkB Pathway:

GenTarget developed a set of reporting lentivirus to monitor NFκB-regulated signaling pathway. Those reporting lentivirus has a luminescent report (Luciferase, Renilla Luc) or a fluorescent report (GFP, RFP) or a  secreted SEAP report, under a minimal CMV promoter (mCMV) that embedded with optimized tandem repeats of NFκB transcriptional response element (NFkB-TRE) sequence motif (5′- GGGACTTTCC). After cells are treated by pro-inflammatory cytokines or stimulants of lymphokine receptors, the NF-κB transcription factors will bind to NFkB-TRE, which activates the minimal promoter, resulted in the downstream reporter’s expression. This pathway lentivirus can be verified by recombinant human tumor necrosis factor alpha (hTNFα) protein.

The luciferase signal can be measured via Luciferase assay. The fluorescent reporter can be detected via its fluorescent signal. The SEAP secreted into cell culture supernatant, which allows to determine reporter activity without disturbing the cells, does not require the preparation of cell lysates and can be used for kinetic studies.

Use Report Lentivirus for NFkB Pathway:

Those reporting lentivirus also contains a constitutively expressed fluorescent selection marker or an antibiotic selection marker under the RSV promoter, which makes it easier to select the stably infected signal reporting cells (to generate pathway specific sensor cell lines), or  provides internal reference for virus transduction efficiency when a fluorescent marker is under the RSV promoter. See lentivector core scheme below.

The premade, ready-to-use reporter lentivirus provides a much easier tool to monitor the activity of NFkB signaling pathways in virtually any mammalian cell type. It also allows to generate your own reporting cell line in your desired cell type for study or screen of pathway specific gene-knockdown, over-expression, or chemical / drug/protein treatment in the cell based assay.

Click the Product Manual (pdf) for all available products for this pathway reporter.