The tumor necrosis factor (TNF) superfamily of cytokines represents a multifunctional group of pro-inflammatory cytokines which activate signaling pathways for cell survival, apoptosis, inflammatory responses, and cellular differentiation. Induction of cellular responses to tumor necrosis factor occurs through two receptors, TNFR1 (TNF Receptor-1 or CD120a) and TNFR2 (TNF Receptor-2 or CD120b). TNFR1 is activated in most human tissues by the binding of TNFα. TNFR2 is expressed in immune cells and is activated by both TNFα and TNFβ [1]. Thermo Scientific™ has a wide range of products to help with TNF research.

Key TNF Pathway Targets

TNF interacts with many pathway targets, including:

AP-1 Caspase 9 NFκB
APAF1 CIAP NIK
ATF Cytochrome c p38
BID ELK1 RAIDD
tBID ERK RIP
c-FOS FADD SODD
c-Jun IκB TAK1
Caspase 1 IKK TRADD
Caspase 2 ITRAF TRAF2
Caspase 3 JNK1 TRAF3
Caspase 6 MADD XIAP
Caspase 7 MAPK  
Caspase 8 MEKK  

In response to inflammation and infection, the pro-inflammatory cytokine TNFα is produced by macrophages, lymphocytes, fibroblasts and keratinocytes. TNFα elicits its pro-inflammatory signals by initially binding to receptors, TNFR1 (p55) and TNFR2 (p75), on the cell surface. TNFR1 and TNFR2 elicit cellular responses to TNFα via three distinct signaling pathways leading to the activation of caspases and the activation of AP-1 and NFκB transcription factors [2]. Activation by TNFα results in the trimerization of the TNFR1 receptor and association of death domains located on the cytoplasmic region of the TNFR1 protein.

Aggregation of the death domains leads to the recruitment of the TRADD (TNFR-Associated Death Domain) adaptor proteins. Additional TRADD mediated recruitment of FADD (Fas-Associated Death Domain), RAIDD (RIP-Associated ICH-1/CED-3-homologous protein with a Death Domain), MADD (MAPK Activating Death Domain) and RIP (Receptor-Interacting Protein) results in the activation of caspase 8. Caspase 8 mediates a series of regulated protein cleavage events that ultimately results in apoptosis.

Cleavage of BID (BH3 interacting death domain) by caspase 8 generates tBID (truncated BID) resulting in the disruption of the mitochondrial membrane and the release of cytochrome c (CytoC). Binding of CytoC to APAF1 (apoptotic protease activating factor-1), allows recruitment of caspase 9 which cleaves caspase 3 creating its active form. The activation of caspases at this stage of the signaling pathway can be inhibited by XIAP (X-linked inhibitor of apoptosis).

Activation of the transcription factors AP-1 and NFκB via TNF signaling is mediated by the association of TRAF2 (TNF Receptor-Associated Factor-2) with TRADD, thereby creating a fully activated complex with MADD, and RIP. Translocation of NFκB to the nucleus occurs when this complex phosphorylates IκK following association with NIK (NF-κB-inducing kinase), resulting in the degradation of IκB [3]. Activation of the AP-1 transcriptional complexes occurs via the activation MAPKs by the TNFR1, TRADD, TRAF2, MADD, RIP complex (Jun NH2-terminal kinase). AP-1 is required for the transcriptional activation of many stress and growth related genes [4].

Data

Thermo Scientific™ offers antibodies, ELISAs, Luminex® multiplex assays and growth factors for key targets in the TNF signaling pathway.

Featured below is ELISA and flow cytometry data using Thermo Scientific™ products.

TNF-alpha_Antibody_PBOTNFAI_ELISA_1_20141103090101
ELISA analysis of TNF-alpha was performed by coating 100 µL per well of anti-bovine TNF-alpha polyclonal antibody (Product # PBOTNFAI) at a concentration of 10 µg/mL and incubating overnight at 4C. The plate was blocked for one hour using 3% bovine serum albumin (BSA) diluted in PBST. The plate was washed six times with 200 µL/well PBST, and then incubated with 100 µL per well of recombinant bovine TNF-alpha protein standards and recombinant ovine TNF-alpha in tissue culture supernatant (both Log2 dilutions starting at an unknown concentration) for one hour at room temperature. The plate was washed with wash buffer six times and incubated with 100 µL per well anti-bovine TNF-alpha biotin-conjugated polyclonal antibody (Product # PBOTNFABI) at 1 µg/mL for one hour at room temperature. The plate was washed with wash buffer six times and then the signal was amplified using HRP-conjugated strepavidin at 2 µg/mL for one hour at room temperature. Detection was performed using 100 µL per well 3,3',5,5'-tetramethylbenzidine (TMB) for 10 minutes. The reaction was stopped with 0.2M sulfuric acid. Absorbance in optical density (OD) values were read on a spectrophotometer at 450nm.
MCD0401-FIX-Perm
C57BL/6 splenocytes were left unstimulated or stimulated for 5 hours with phorbol myristate acetate (PMA) and ionomycin in the presence of brefeldin A. Cells were then surface-stained with anti-mouse CD4 FITC (Product # MCD0401) and subsequently fixed and permeabilized using the FIX & PERM® Cell Permeabilization Kit (Product # GAS003 or GAS004) and stained intracellularly with anti-mouse interferon gamma (IFN-gamma) PE-Cy®7 conjugate (Product # A18713) and anti-mouse tumor necrosis factor alpha (TNF-a) Pacific Blue™ conjugate (Product # RM90128). Samples were collected using the Attune® Acoustic Focusing Cytometer (blue/violet) with 488 nm excitation and 530/30 nm bandpass emission filter to detect FITC, and 640 nm longpass filter to detect PE-Cy®7 conjugate. 405 nm excitation and 450/40 nm bandpass emission filter were used to detect the Pacific Blue™ fluorescence. PANEL A: IFN-gamma and TNF-a co-staining of total mouse splenocytes that were left unstimulated (left panel) or stimulated (right panel) with PMA and ionomycin in the presence of brefeldin A. PANEL B: CD4 T cell expression of TNF-a (left panel) and IFN-gamma (right panel) after stimulation with the above stated conditions.

References

  1. Kawasaki H. et al. (2002) Identification of genes that function in the TNFα-mediated apoptotic pathway using randomized hybrid ribozyme libraries. Nat Biotechnol 20: 376-80.
  2. Kagoya, Y. et al. (2014) Positive feedback between NF-κB and TNF-α promotes leukemia-initiating cell capacity. J Clin Invest 124(2): 528-542.
  3. Baud V. et al. (2001) Signal transduction by tumor necrosis factor and its relatives. Trends Cell Biol 11: 372-7.
  4. Kai-Li He. et al. (2002) A20 Inhibits Tumor necrosis factor (TNF) α-Induced apoptosis by disrupting recruitment of TRADD and RIP to the TNF receptor 1 complex in jurkat T cells. Mol Cell Biol 22: 6034-6045.


Product Name Catalog Number
c-Myc Antibody 132500
Cytochrome C Antibody 456100
Caspase 3 Antibody PA516335
RIPK1 Antibody
PA520811
c-Fos Antibody
PA1830
TAK1/MAP3K7 Antibody
PA517507
BID [p15] Antibody
44433G
XIAP Antibody PA529253
MEKK1/MAP3K1 Antibody PA515085
p38 Antibody PA527831
ELK1 Antibody
458700
Caspase 1 Antibody
PA529342
Caspase 8 Antibody
PA137242
TRAF2 Antibody
PA529238
TRAF3 Antibody
PA141107
cIAP Antibody
PA520066
FADD Antibody
PA517808
TRADD Antibody
PA521595
Apaf1 Antibody PA519893