2B4/CD244/SLAMF4 Overexpression Lysate

Western Blot: 2B4/CD244/SLAMF4 Overexpression Lysate [NBL1-08924] - Left-Empty vector transfected control cell lysate (HEK293 cell lysate); Right -Over-expression Lysate for CD244.

• Reactivity: Hu
• Applications: WB

2B4/CD244/SLAMF4 Overexpression Lysate Summary

• Description:

  2B4/CD244/SLAMF4 Transient Overexpression Lysate

  
  Expression Host: HEK293T
  
  Plasmid: RC206988
  
  Accession#: NM_016382
  
  Protein Tag: C-MYC/DDK
  
  You will receive 1 vial of lysate (100ug), 1 vial of empty vector negative control (100ug), and 1 vial of 2xSDS sample buffer (250ul). Each vial of cell lysate contains 100ug of total protein (at 1 mg/ml). The 2xSDS Sample Buffer consists of 4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromophenol blue, 100mM DTT.
• Gene: CD244

Applications/Dilutions

• Dilutions:
  • Western Blot
• Application Notes: This product is intended for use as a positive control in Western Blot. Overexpression of the target protein was confirmed using an antibody to DDK (FLAG) epitope tag (NBP1-71705) present on the protein construct.
  
  Each vial of cell lysate contains 100ug of total protein which should be sufficient for 20-50 reactions. Depending on over-expression level, antibody affinity and detection system, some lysates can go as low as 0.1 ug per load. We recommend starting with 5ug of cell lysate. Add an equal amount of cell lysate and 2X SDS Sample buffer and boil the SDS samples for 10 minutes before loading.
• Theoretical MW: 39 kDa.
  Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.

Packaging, Storage & Formulations

• Storage: Store at -80C. Avoid freeze-thaw cycles.
• Buffer: RIPA buffer

Lysate Details for Array

• Type: Overexpression

Notes

HEK293T cells in 10-cm dishes were transiently transfected with a non-lipid polymer transfection reagent specially designed and manufactured for large volume DNA transfection. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4, and then centrifuged to clarify the lysate. Protein concentration was measured by BCA protein assay kit.

Alternate Names for 2B4/CD244/SLAMF4 Overexpression Lysate

• 2B4
• CD244 antigen
• CD244 molecule, natural killer cell receptor 2B4
• CD244
• h2B4
• NAIL
• NAILNK cell activation-inducing ligand
• natural killer cell receptor 2B4
• NKR2B4
• NKR2B4NK cell type I receptor protein 2B4,2B4CD244 natural killer cell receptor 2B4
• Nmrk
• SLAMF4
• SLAMF4NK cell activation inducing ligand NAIL

Background

2B4, also known as CD244 and SLAMF4, is a type I transmembrane glycoprotein that is member of the signaling lymphocyte activation molecule (SLAM) subfamily in immunoglobulin (Ig) superfamily (1,2). The 2B4 protein plays a role in immune system associated diseases including the recognition and clearance of cancer cells, infected cells, and key allergy cells (1). 2B4 is expressed as a heterophilic receptor on the cell membrane of natural killer (NK) cells in addition to on monocytes and basophils, gamma/delta T cells, a subset of CD8+ T cells, and other immune cells and tissues (1). Additionally, 2B4 is considered a marker for CD8+ T cell exhaustion in chronic infection and increased expression has been observed in diseases including lymphocytic choriomeningitis virus (LCMV) and hepatitis B virus (HBV) (2). Human 2B4 protein is 370 amino acids (aa) in length with a theoretical molecular weight (MW) of 41 kDa (3). However, 2B4 contains multiple glycosylation sites that increases its observed MW to ~70 kDa (3). The 2B4/CD244 protein consists of a 21 aa signal peptide followed by an extracellular domain (ECD) with two Ig-like domains, a single transmembrane domain, and four cytoplasmic immunoreceptor tyrosine-based switch motifs (ITSMs) (3).

The 2B4 receptor has both activating and inhibitory signaling properties (1,4). 2B4's ligand is CD48, which is expressed on hematopoietic cells (4, 5). When bound to CD48, subsequent phosphorylation of the ITSMs occurs, followed by recruitment of SH-2-containing adaptor protein (SAP) and Fyn, leading to phosphorylation of PLCgamma and Vav1, and resulting in NK cell activation (4). In the absence of SAP, Src homology region 2-containing protein tyrosine phosphatases (SHP) get recruited and inhibits NK cell activation (4). In the context of infection such as Epstein-Barr virus (EBV), when lacking SAP, 2B4 has been shown to have an inhibitory function via impaired T cell cytotoxicity against EBV-infected cells (6). While 2B4 has a role in eliciting CD8+ T cell response, overexpression of 2B4 can lead to T cell exhaustion in states of chronic infection (6). Studies have suggested that the antibody blockade of the 2B4-CD48 interaction might enhance T cell and NK cell function, however given both the activating and inhibitory properties of the 2B4 receptor, more research needs to be done to prevent unwanted effects (6).

References

1. Buller CW, Mathew PA, Mathew SO. Roles of NK Cell Receptors 2B4 (CD244), CS1 (CD319), and LLT1 (CLEC2D) in Cancer. Cancers (Basel). 2020 Jul 1;12(7):1755. https://doi.org/10.3390/cancers12071755

2. Catakovic K, Klieser E, Neureiter D, Geisberger R. T cell exhaustion: from pathophysiological basics to tumor immunotherapy. Cell Commun Signal. 2017;15(1):1. https://doi.org/10.1186/s12964-016-0160-z

3. Uniprot (Q9BZW8)

4. Chen Y, Lu D, Churov A, Fu R. Research Progress on NK Cell Receptors and Their Signaling Pathways. Mediators Inflamm. 2020 Jul 24;2020:6437057. https://doi.org/10.1155/2020/6437057.

5. Pahima H, Puzzovio PG, Levi-Schaffer F. 2B4 and CD48: A powerful couple of the immune system. Clin Immunol. 2019 Jul;204:64-68. https://doi.org/10.1016/j.clim.2018.10.014.

6. Waggoner SN, Kumar V. Evolving role of 2B4/CD244 in T and NK cell responses during virus infection. Front Immunol. 2012;3:377. https://doi.org/10.3389/fimmu.2012.00377

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: CD244