近红外双荧光系统在磷酸酯酶筛选中的应用
|
摘要
目的应用奥德赛(Odyssey)近红外双荧光系统筛选去甲基化酶KDM3A去磷酸化的磷酸酯酶。方法使用磷酸酯酶干扰质粒转染HEK293T细胞,使用磷酸化的KDM3A抗体对细胞进行杂交,近红外荧光染料DRAQ5标记细胞核,利用奥德赛(Odyssey)近红外双荧光系统对细胞进行荧光扫描,荧光强度的比值可代表细胞内相对KDM3A磷酸化水平,进而筛选出相关的磷酸酯酶。结果磷酸酯酶广谱抑制剂冈崎酸OA处理转染后的细胞,近红外双荧光扫描系统可以检测到KDM3A的磷酸化,初步筛选出4个可能参与KDM3A去磷酸化的磷酸酯酶亚基。结论近红外双荧光系统可以应用于磷酸酯酶的初步筛选。
Objective To determine the phosphates of histone demethylase KDM3A with the application of Odyssey dual-infrared fluorescence system.Methods HEK293T cells had been transfected with phosphates interfere plasmids.After incubating with phosphorylation-KDM3A antibody and infrared fluorescent nuclei dyes DRAQ5,HEK293T cells were scanned with Odyssey dual-infrared fluorescence scan system.The ratio of fluorescence intensity could represent the relative KDM3A phosphorylation level in vivo.Results Okazaki acid(OA),the broad-spectrum inhibitor of phosphates,was applied to treat the cells.The signals were detected for phosphorylated KDM3A by dual-infrared fluorescence scan system.Four phosphate subunits that may participate in the dephosphorylation of KDM3A were determined by Odyssey dual-infrared fluorescence scan system.Conclusion Odyssey dual-infrared fluorescence scan system was able to be applied in the screening for protein phosphotases.
Objective To determine the phosphates of histone demethylase KDM3A with the application of Odyssey dual-infrared fluorescence system.Methods HEK293T cells had been transfected with phosphates interfere plasmids.After incubating with phosphorylation-KDM3A antibody and infrared fluorescent nuclei dyes DRAQ5,HEK293T cells were scanned with Odyssey dual-infrared fluorescence scan system.The ratio of fluorescence intensity could represent the relative KDM3A phosphorylation level in vivo.Results Okazaki acid(OA),the broad-spectrum inhibitor of phosphates,was applied to treat the cells.The signals were detected for phosphorylated KDM3A by dual-infrared fluorescence scan system.Four phosphate subunits that may participate in the dephosphorylation of KDM3A were determined by Odyssey dual-infrared fluorescence scan system.Conclusion Odyssey dual-infrared fluorescence scan system was able to be applied in the screening for protein phosphotases.
引文
1 Klose RJ,Kallin EM,Zhang Y.JmjC-domain-containing proteins and histone demethylation[J].Nat Rev Genet,2006,7(9):715-727
2 Okada Y.Histone demethylase JHDM2A is critical for Tnp1 and Prm1transcription and spermatogenesis[J].Nature,2007,450(7166):119-123
3 Okada Y,Tateishi K,Zhang Y.Histone demethylase JHDM2A is involved in male infertility and obesity[J].J Androl,2010,31(1):75-78
4 Krieg AJ.Regulation of the histone demethylase JMJD1A by hypoxiainducible factor 1 alpha enhances hypoxic gene expression and tumor growth[J].Mol Cell Biol,2010,30(1):344-353
5 Goris J.Okadaic acid,a specific protein phosphatase inhibitor,induces maturation and MPF formation in Xenopus laevis oocytes[J].FEBS Lett,1989,245(1-2):91-94
6 Franchini A,Malagoli D,Ottaviani E.Targets and effects of yessotoxin,okadaic acid and palytoxin:a differential review[J].Mar Drugs,2010,8(3):658-677
7 Yamane K.JHDM2A,a JmjC-containing H3K9 demethylase,facilitates transcription activation by androgen receptor[J].Cell,2006,125(3):483-495
8 Yamada D.Role of the hypoxia-related gene,JMJD1A,in hepatocellular carcinoma:clinical impact on recurrence after hepatic resection[J].Ann Surg Oncol,2012,19 Suppl 3:S355-364
9 Pedersen MT,Helin K.Histone demethylases in development and disease[J].Trends Cell Biol,2010,20(11):662-671
10 Cho HS.The JmjC domain-containing histone demethylase KDM3A is a positive regulator of the G1/S transition in cancer cells via transcriptional regulation of the HOXA1 gene[J].Int J Cancer,2012,131 (3):E179-189
11 Inagaki T.Obesity and metabolic syndrome in histone demethylase JHDM2a-deficient mice[J].Genes Cells,2009,14(8):991-1001
12 Ma DK.G9a and Jhdm2a regulate embryonic stem cell fusion-induced reprogramming of adult neural stem cells[J].Stem Cells,2008,26(8):2131-2141
2 Okada Y.Histone demethylase JHDM2A is critical for Tnp1 and Prm1transcription and spermatogenesis[J].Nature,2007,450(7166):119-123
3 Okada Y,Tateishi K,Zhang Y.Histone demethylase JHDM2A is involved in male infertility and obesity[J].J Androl,2010,31(1):75-78
4 Krieg AJ.Regulation of the histone demethylase JMJD1A by hypoxiainducible factor 1 alpha enhances hypoxic gene expression and tumor growth[J].Mol Cell Biol,2010,30(1):344-353
5 Goris J.Okadaic acid,a specific protein phosphatase inhibitor,induces maturation and MPF formation in Xenopus laevis oocytes[J].FEBS Lett,1989,245(1-2):91-94
6 Franchini A,Malagoli D,Ottaviani E.Targets and effects of yessotoxin,okadaic acid and palytoxin:a differential review[J].Mar Drugs,2010,8(3):658-677
7 Yamane K.JHDM2A,a JmjC-containing H3K9 demethylase,facilitates transcription activation by androgen receptor[J].Cell,2006,125(3):483-495
8 Yamada D.Role of the hypoxia-related gene,JMJD1A,in hepatocellular carcinoma:clinical impact on recurrence after hepatic resection[J].Ann Surg Oncol,2012,19 Suppl 3:S355-364
9 Pedersen MT,Helin K.Histone demethylases in development and disease[J].Trends Cell Biol,2010,20(11):662-671
10 Cho HS.The JmjC domain-containing histone demethylase KDM3A is a positive regulator of the G1/S transition in cancer cells via transcriptional regulation of the HOXA1 gene[J].Int J Cancer,2012,131 (3):E179-189
11 Inagaki T.Obesity and metabolic syndrome in histone demethylase JHDM2a-deficient mice[J].Genes Cells,2009,14(8):991-1001
12 Ma DK.G9a and Jhdm2a regulate embryonic stem cell fusion-induced reprogramming of adult neural stem cells[J].Stem Cells,2008,26(8):2131-2141