DNMT3A 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.

Methylation of cytosine residues is essential for mammalian development by regulating gene expression, gene silencing and genomic imprinting (1). This process is controlled by the enzyme DNA (cytosine-5)-methyltransferase 3A (DNMt3a), which is encoded by the DNMT3A gene and has a predicted molecular weight of 102 kDa. DNA methyltransferases including DNMt3a are involved in de novo cytosine methylation and maintenance of embryonic and somatic cells through the transfer of methyl groups to specific CpG structures in DNA (2). Somatic mutations in DNMT3A are responsible for cytogenetically normal acute myeloid leukemia (CN-AML). Hypermethylation of CpG islands in tumor suppressor genes or hypomethylation of bulk genomic DNA are linked with cancer (3,4). Mutations in the DNMT3A gene have also been found to cause DNMT3A overgrowth syndrome and myelodysplastic syndromes.

References

1. Thakur, A., Mackin, S. J., Irwin, R. E., O'Neill, K. M., Pollin, G., & Walsh, C. (2016). Widespread recovery of methylation at gametic imprints in hypomethylated mouse stem cells following rescue with DNMT3A2. Epigenetics Chromatin, 9, 53. doi:10.1186/s13072-016-0104-2

2. Ravichandran, M., Lei, R., Tang, Q., Zhao, Y., Lee, J., Ma, L., . . . Dawlaty, M. M. (2019). Rinf Regulates Pluripotency Network Genes and Tet Enzymes in Embryonic Stem Cells. Cell Rep, 28(8), 1993-2003.e1995. doi:10.1016/j.celrep.2019.07.080

3. Pang, Y., Liu, J., Li, X., Xiao, G., Wang, H., Yang, G., . . . Ren, H. (2018). MYC and DNMT3A-mediated DNA methylation represses microRNA-200b in triple negative breast cancer. J Cell Mol Med, 22(12), 6262-6274. doi:10.1111/jcmm.13916

4. Xia, L., Huang, W., Bellani, M., Seidman, M. M., Wu, K., Fan, D., . . . Baylin, S. B. (2017). CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes. Cancer Cell, 31(5), 653-668.e657. doi:10.1016/j.ccell.2017.04.005

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 NBL1-09970

• DNMT3A Antibodies
• DNMT3A ELISA Kits
• DNMT3A Kit
• DNMT3A Lysate
• DNMT3A Proteins