Kinase preparations and assays: GSK-3β is assayed, following a 1/100 dilution in 1 mg BSA per mL 10 mM dithiothreitol, with 5 μL 40 μM GS-1 peptide as a substrate, in buffer A, in the presence of 15 μM [γ-32P]ATP (3000 Ci·mmol-1; 1 mCi·mL-1 ) in a final volume of 30 μL. After 30 min incubation at 30℃, 25 μL aliquots of supernatant are spotted onto 2.5×3 cm pieces of Whatman P81 phosphocellulose paper, and 20 s later, the filters are washed five times in a solution of 10 mL phosphoric acid per L of water. The wet filters are counted in the presence of 1 mL ACS scintillation fluid. The kinase activity of CDK1/cyclin B is assayed in buffer C, with 1 mg/mL histone H1, in the presence of 15 μM [γ-32P]ATP (3000 Ci·mmol-1; 1 mCi·mL-1 ) in a final volume of 30 μL. After 10 min incubation at 30℃, 25 μL aliquots of supernatant are spotted onto P81 phosphocellulose papers and treated as described above. The activity of CDK5/p25 is assayed in buffer C as described for CDK1/cyclin B. (Buffer A: 10 mM MgCl2 , 1 mM EGTA, 1 mM dithiothreitol, 25 mM Tris/HCl pH 7.5, 50 μg heparin/mL. Buffer C: homogenization buffer but 5 mM EGTA, no NaF and no protease inhibitors.)[6]
[1]. Christman JK. 5-Azacytidine and 5-aza-2′-deoxycytidine as inhibitors of DNA methylation: mechanistic studies and their implications for cancer therapy. Oncogene. 2002 Aug 12; 21 (35) :5483-95.
[2]. Creusot F, et al. Inhibition of DNA methyltransferase and induction of Friend erythroleukemia cell differentiation by 5-azacytidineand 5-aza-2′-deoxycytidine. J Biol Chem. 1982 Feb 25; 257 (4) :2041-8.
[3]. Li LH, et al. Cytotoxicity and mode of action of 5-azacytidine on L1210 leukemia. Cancer Res. 1970 Nov; 30 (11) :2760-9.
备注
以上数据均来自公开文献, Jinpan暂未进行独立验证, 仅供参考。These protocols are for reference only. Jinpan does not independently validate these methods.
规格
10mg 50mg 10mM*1mL (in DMSO) 100mg
为一种低甲基化的强效生长抑制剂及细胞毒素剂，抑制 DNA 甲基转移酶。也是作用于S期的细胞周期特异性化合物，能迅速磷酸化并掺入RNA，通过破坏核酸顺利翻译为蛋白质，而抑制蛋白质的合成。还可通过抑制乳清酸核苷酸脱羧酶而影响嘧啶的合成。是一种基因表达、基因激活和沉默的重要调节剂。是胞苷的核苷类似物，其通过捕获 DNA甲基转移酶 特异性地抑制DNA甲基化。
1]. Nakamura M, et al. Decitabine inhibits tumor cell proliferation and up-regulates E-cadherin expression in Epstein-Barr virus-associated gastric cancer. J Med Virol. 2016 Jul 19. [2]. Zhang C, et al. Quantitative proteomic analysis of histone modifications in decitabine sensitive and resistant leukemia cell lines. Clin Proteomics. 2016 Jul 5;13:14. [3]. Requena CE, et al. The nucleotidohydrolases DCTPP1 and dUTPase are involved in the cellular response to decitabine. Biochem J. 2016 Jun 20. [4]. Terse P, et al. Subchronic oral toxicity study of decitabine in combination with tetrahydrouridine in CD-1 mice. Int J Toxicol. 2014 Mar-Apr;33(2):75-85. [5]. Yu J, et al. DNA methyltransferase expression in triple-negative breast cancer predicts sensitivity to decitabine. J Clin Invest. 2018 Jun 1;128(6):2376-2388.
