CD63 Overexpression Lysate

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.

CD63 (cluster of differentiation 63), also known as lysosome associated membrane protein 3 (LAMP-3), is a membrane spanning glycoprotein with a molecular mass ranging from 30-60 kDa that was the first characterized member of the tetraspanin superfamily (1,2). CD63 is ubiquitously expressed and largely present on the cell surface in the endosomal system (1-3). More specifically, it is generally present in multivesicular bodies (MVBs), also called late endosomes, and lysosomes (1). The CD63 molecule is a total of 238 amino acids (aa), has four hydrophobic membrane-spanning regions and three N-glycosylation sites, and the encoded protein has a theoretical molecular weight of 25 kDa (2,3). CD63 both directly and indirectly interacts with other proteins including integrins, cell surface receptors, other tetraspanins, kinases, and adapter proteins, to name a few (1). CD63 is involved in many cell processes including cell survival and activation, cell adhesion, invasion, and migration (1). Additionally, CD63 has been shown to interact with tissue inhibitor of metalloproteinase 1 (TIMP-1), which originally thought to be an inhibitor of cancer progression has recently been shown to have cancer promoting properties as well (1,4). The CD63-TIMP-1 interaction has been shown to activate the PI3K/AKT pathway in lung adenocarcinoma cells and also promote survival and invasion of acute myeloid leukemia cells (1).CD63 is a useful flow cytometry marker for basophil granulocytes and can be used for the basophil activation test (BAT) to assess IgE-mediated allergy response (5). As CD63 was initially discovered on activated blood platelets and is a lysosomal-associated protein it makes sense it would be involved in platelet or lysosomal-related disorders (1,2). More precisely, CD63 is associated with Hermansky-Pudlak syndrome, a disease characterized by albinism and platelet storage pool deficiency (6).

References

1. Pols, M. S., & Klumperman, J. (2009). Trafficking and function of the tetraspanin CD63. Experimental cell research. https://doi.org/10.1016/j.yexcr.2008.09.020

2. Metzelaar, M. J., Wijngaard, P. L., Peters, P. J., Sixma, J. J., Nieuwenhuis, H. K., & Clevers, H. C. (1991). CD63 antigen. A novel lysosomal membrane glycoprotein, cloned by a screening procedure for intracellular antigens in eukaryotic cells. The Journal of biological chemistry.

3. Horejsi, V., & Vlcek, C. (1991). Novel structurally distinct family of leucocyte surface glycoproteins including CD9, CD37, CD53 and CD63. FEBS letters. https://doi.org/10.1016/0014-5793(91)80988-f

4. Eckfeld, C., HauBler, D., Schoeps, B., Hermann, C. D., & Kruger, A. (2019). Functional disparities within the TIMP family in cancer: hints from molecular divergence. Cancer metastasis reviews. https://doi.org/10.1007/s10555-019-09812-6

5. Hoffmann, H. J., Santos, A. F., Mayorga, C., Nopp, A., Eberlein, B., Ferrer, M., Rouzaire, P., Ebo, D. G., Sabato, V., Sanz, M. L., Pecaric-Petkovic, T., Patil, S. U., Hausmann, O. V., Shreffler, W. G., Korosec, P., & Knol, E. F. (2015). The clinical utility of basophil activation testing in diagnosis and monitoring of allergic disease. Allergy. https://doi.org/10.1111/all.12698

6. Dell'Angelica, E. C., Shotelersuk, V., Aguilar, R. C., Gahl, W. A., & Bonifacino, J. S. (1999). Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the beta 3A subunit of the AP-3 adaptor. Molecular cell. https://doi.org/10.1016/s1097-2765(00)80170-7

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.

Array NBL1-08960

• CD63 Antibodies
• CD63 ELISA Kit
• CD63 Lysate
• CD63 Proteins
• CD63 Proteins and Enzyme