CD28 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.

CD28 (cluster differentiation 28) is a 44 kDa disulfide linked homodimeric T cell specific surface glycoprotein with a role in providing co-stimulatory signals required for T cell activation and survival (1). The CD28 family of receptors, including PD-1, CTLA-4, and ICOS, share several common features including paired V-set immunoglobulin superfamily (IgSF) domains attached to a single transmembrane domain and cytoplasmic domains containing critical signaling motifs (2). Additionally, CD28 and CTLA-4 are very similar in genomic organization. The corresponding genes co-map on human chromosome 2q33 and mouse chromosome 1 (3). Human CD28 isoform 1 is synthesized as a protein of 220 amino acids (aa) in length with a calculated molecular weight of 25 kDa (3).

CD28 is the prototypical and best-characterized costimulatory molecule on T cells (4). Its signals are critical for optimal naive T cell activation, cytokine production, proliferation, and survival (4). In order to sustain T cell activation, CD28 will consolidate immunological synapse formation, increase cell cycle progression through upregulated D-cyclin expression, and aid in T cell survival by in inducing the expression of the anti-apoptotic protein Bcl-XL (5). CD28 is constitutively expressed on naive and central memory CD4+ and CD8+ cells (5). CD28 deficiency has a large impact on T cell responses including activation, proliferation, immunoglobulin (Ig) class-switching, and germinal center (GC) formation (6). CD28 is a critical regulator of autoimmune diseases and tolerance to solid organ transplants in human patients (6). The CD28 pathway plays a central role in immune responses against pathogens, autoimmune diseases, and graft rejection (7). CD28 engagement via antibodies augments the proliferation of T cells in response to immobilized anti-CD3 antibodies (8). Additionally, antibody engagement of CD28 can supply costimulation to T cells encountering APCs deficient in costimulatory ligands, such as CD80 and CD86, and prevents the resultant anergic state that otherwise occurs in the absence of costimulatory signaling (8).

References

1. Esensten, J. H., Helou, Y. A., Chopra, G., Weiss, A., & Bluestone, J. A. (2016). CD28 Costimulation: From Mechanism to Therapy. Immunity, 44(5), 973-988. https://doi.org/10.1016/j.immuni.2016.04.020

2. Carreno, B. M., & Collins, M. (2002). The B7 family of ligands and its receptors: new pathways for costimulation and inhibition of immune responses. Annual review of immunology, 20, 29-53. https://doi.org/10.1146/annurev.immunol.20.091101.091806

3. Ward S. G. (1996). CD28: a signaling perspective. The Biochemical journal, 318 (Pt 2), 361-377. https://doi.org/10.1042/bj3180361

4. Zhang, R., Huynh, A., Whitcher, G., Chang, J., Maltzman, J. S., & Turka, L. A. (2013). An obligate cell-intrinsic function for CD28 in Tregs. The Journal of clinical investigation, 123(2), 580-593. https://doi.org/10.1172/JCI65013

5. Evans, E. J., Esnouf, R. M., Manso-Sancho, R., Gilbert, R. J., James, J. R., Yu, C., Fennelly, J. A., Vowles, C., Hanke, T., Walse, B., Hunig, T., Sorensen, P., Stuart, D. I., & Davis, S. J. (2005). Crystal structure of a soluble CD28-Fab complex. Nature immunology, 6(3), 271-279. https://doi.org/10.1038/ni1170

6. Bour-Jordan, H., & Blueston, J. A. (2002). CD28 function: a balance of costimulatory and regulatory signals. Journal of clinical immunology, 22(1), 1-7. https://doi.org/10.1023/a:1014256417651

7. Krummel, M. F., & Allison, J. P. (1995). CD28 and CTLA-4 have opposing effects on the response of T cells to stimulation. The Journal of experimental medicine, 182(2), 459-465. https://doi.org/10.1084/jem.182.2.459

8. Luhder, F., Huang, Y., Dennehy, K. M., Guntermann, C., Muller, I., Winkler, E., Kerkau, T., Ikemizu, S., Davis, S. J., Hanke, T., & Hunig, T. (2003). Topological requirements and signaling properties of T cell-activating, anti-CD28 antibody superagonists. The Journal of experimental medicine, 197(8), 955-966. https://doi.org/10.1084/jem.20021024

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 NBP2-04308

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