Fas/TNFRSF6/CD95 Knockout HeLa Cell Lysate

Product Discontinued

Fas/TNFRSF6/CD95 Knockout HeLa Cell Lysate Summary

• Preparation Method: Knockout achieved by using CRISPR/Cas9,1 bp insertion in exon2
• Gene: FAS

Applications/Dilutions

• Dilutions:
  • Western Blot
• Application Notes: You will receive 1 vial (100ug) of knockout cell lysate and 1 vial (100ug) of Parental cell lysate. Lysate can be diluted with 1X SDS sample buffer and will be stable at -20 degrees C for 12 months. Minimize freeze-thaw cycles.

Packaging, Storage & Formulations

• Storage: Store at -20C short term. Aliquot and store at -80C long term. Avoid freeze-thaw cycles.
• Buffer: 0.1 mg cell homogenate lyophilized in RIPA buffer made with double-knockout cell lines.
• Concentration: LYOPH
• Reconstitution Instructions: To use as WB negative control, spin down briefly and resuspend in 100 uL 1xSDS sample buffer (2% SDS, 60 mM Tris-HCl pH 6.8, 10% Glycerol, 0.02% Bromophenol blue, 60 mM beta-mercaptoethanol). Boil the lysate for 3 - 5 minutes before loading it onto gel.

Lysate Details for Array

• Type: Knockout HeLa Cell

Notes

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Validation of antibody specificity is critical and verification of antibody performance against knockout samples is one way to guarantee that an antibody recognizes a specific target. Novus' KO cell lysate can be used as a negative control for western blots and to confirm the specificity of antibodies.

Alternate Names for Fas/TNFRSF6/CD95 Knockout HeLa Cell Lysate

• Apo-1 antigen
• Apo-1
• apoptosis antigen 1
• Apoptosis-mediating surface antigen FAS
• APT1
• APT1FASTM
• CD95 antigen
• CD95
• CD95ALPS1A
• Fas (TNF receptor superfamily, member 6)
• Fas AMA
• Fas antigen
• Fas
• FAS1
• FASLG receptor
• TNFRSF6
• TNFRSF6member 6
• tumor necrosis factor receptor superfamily member 6

Background

Tumor Necrosis Family Receptor (TNFR) superfamily member Fas, also known as CD95, APO-1, and TNFRSF6, is a 40-50 kDa type I transmembrane glycoprotein that is traditionally considered a death receptor but also functions in non-apoptotic signaling (1-4). The human Fas/TNFRSF6/CD95 protein is encoded by the FAS gene which contains 9 exons and is located on chromosome 10 (10q23.3-4) (1,2). The mature canonical Fas/TNFRSF6 protein isoform is 335 aa in length, which includes the signal sequence, and has a theoretical molecular weight of 37.7 kDa (1,5). The protein contains an extracellular domain (ECD) consisting of three calcium rich domains (CRDs), a transmembrane domain (TM), and an intracellular domain (ICD) comprised of a calcium-inducing domain (CID) and characteristic dead domain (DD) (1,2,5,6). The Fas protein is expressed on the plasma membrane of activated lymphocytes as a homotrimer formed via CRD1 interactions (1,2,3,6). The DD is crucial for apoptotic signaling which is triggered by the Fas receptor binding its ligand, Fas ligand (FasL) (1,2,6,7). Upon Fas-FasL interaction, the DD recruits an adapter protein Fas-associated DD (FADD) and procaspase-8, generating the death-inducing signaling complex (DISC) (1-4,6-8). Formation of DISC activates caspase-8 and leads to cleavage of caspase-3, initiating a caspase-signaling cascade and cell death (1-4,6-8).

Fas-FasL-mediated apoptosis is important in immune homeostasis and removal of autoreactive T cells, autoreactive B cells, cytotoxic natural killer (NK) cells, and more (1,2,7). Dysfunction and mutations in the Fas receptor and the Fas-FasL signaling axis is associated a loss of apoptotic signaling and removal of autoreactive cells, which correlates with several autoimmune diseases including systemic lupus erythematosus (SLE), autoimmune lymphoproliferative syndrome (ALPS), and multiple sclerosis (MS) (1-4,6,7). In addition to apoptosis and cell death signaling, FasL/TNFRSF6/CD95 mediates other pathways involved in proliferation, survival, and differentiation (3,4,6,8). More specifically, Fas has been shown to activate the NF-kappaB pathway, driving innate immunity which includes IL-1beta production and functioning in host defense (3,4,6,8). Fas is also involved in adaptive immunity playing a role in co-stimulation of CD4+ and CD8+ T cell activation as well as precocious differentiation of naive cells to effector memory T cells (3,4,6). Differentiation into effector memory T cells shows protection against autoimmunity but also limits antitumor response to a form of cancer immunotherapy called adoptive cell transfer (ACT) (3,4). The non-apoptotic roles of the Fas/TNFRSF6/CD95 receptor highlight its potential as a target for both treating autoimmune diseases and in cancer immunotherapy (3,4).

References

1. Singh R, Pradhan V, Patwardhan M, Ghosh K. APO-1/Fas gene: Structural and functional characteristics in systemic lupus erythematosus and other autoimmune diseases. Indian J Hum Genet. 2009;15(3):98-102. https://doi.org/10.4103/0971-6866.60184

2. Magerus A, Bercher-Brayer C, Rieux-Laucat F. The genetic landscape of the FAS pathway deficiencies. Biomed J. 2021;44(4):388-399. https://doi.org/1010.1016/j.bj.2021.06.005

3. Guegan JP, Legembre P. Nonapoptotic functions of Fas/CD95 in the immune response. FEBS J. 2018;285(5):809-827. https://doi.org/10.1111/febs.14292

4. Yi F, Frazzette N, Cruz AC, Klebanoff CA, Siegel RM. Beyond Cell Death: New Functions for TNF Family Cytokines in Autoimmunity and Tumor Immunotherapy. Trends Mol Med. 2018;24(7):642-653. https://doi.org/10.1016/j.molmed.2018.05.004

5. Uniprot (P25445)

6. Guegan JP, Ginestier C, Charafe-Jauffret E, et al. CD95/Fas and metastatic disease: What does not kill you makes you stronger. Semin Cancer Biol. 2020;60:121-131. https://doi.org/10.1016/j.semcancer.2019.06.004

7. Volpe E, Sambucci M, Battistini L, Borsellino G. Fas-Fas Ligand: Checkpoint of T Cell Functions in Multiple Sclerosis. Front Immunol. 2016;7:382. Published 2016 Sep 27. https://doi.org/10.3389/fimmu.2016.00382

8. Cullen SP, Martin SJ. Fas and TRAIL 'death receptors' as initiators of inflammation: Implications for cancer. Semin Cell Dev Biol. 2015;39:26-34. https://doi.org/10.1016/j.semcdb.2015.01.012

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Lysates are guaranteed for 6 months from date of receipt.

Bioinformatics

• Gene Symbol: FAS