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Involvement of ERK1/2 and p38 MAPK in up-regulation of 14-3-3 protein induced by hydrogen peroxide preconditioning in PC12 cells
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作者 苏庆杰 陈小武 +1 位作者 陈志斌 孙圣刚 《Neuroscience Bulletin》 SCIE CAS CSCD 2008年第4期244-250,共7页
Objective To investigate the protective effects of hydrogen peroxide preconditioning (HPP) on the pheochromocytoma (PC12) cells treated with 1-methyl-4-phenylpyridinium (MPP^+) and to explore the potential mech... Objective To investigate the protective effects of hydrogen peroxide preconditioning (HPP) on the pheochromocytoma (PC12) cells treated with 1-methyl-4-phenylpyridinium (MPP^+) and to explore the potential mechanisms. Methods The viability and apoptosis of PC 12 cells were determinded by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 4′,6′-diamidino-2-phenylindole (DAPI) staining, respectively. The expressions of 14-3-3 protein and phospholylated p38 mitogen-activated protein kinase (MAPK) were determined by Western blot. Enzyme-linked immunosorbent assay (ELISA) was used to measure the activity of extracellular signal-regulated protein kinase 1/2 (ERK1/2). Results The cell viability decreased and the number of apoptotic cells increased dramatically in MPP^+ group compared with that in Control group. HPP induced a significant increase in cell viability and a marked decrease in population of apoptotic cells of the MPP^+- treated PC 12 cells, accompanied with up-regulation of 14-3-3 protein and increase of ERK 1/2 and p38 MAPK activities. The 14-3-3 protein expression was positively correlated with the phosphorylation of ERK1/2. Furthermore, inhibition of the ERK1/2 with PD98059 abolished the 14-3-3 protein up-regulation in PC 12 cells induced by HPP. Conclusion HPP protects PC 12 cells against MPP+ toxicity by up-regulating 14-3-3 protein expression through the ERK1/2 and p38 MAPK signaling pathways. 展开更多
关键词 hydrogen peroxide preconditioning 14-3-3 protein ERK1/2 p38 mitogen-activated protein kinase pC12 cell
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GST-P12融合蛋白的抗体制备、鉴定及免疫组化分析 被引量:2
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作者 邵小钧 陈伟 +3 位作者 孙沫逸 孙文斌 戴建武 张圃 《现代肿瘤医学》 CAS 2007年第4期463-465,共3页
目的:为了进一步研究p12DOC-1基因及其蛋白在口腔癌发生中的作用与调控机制,将已表达纯化好的GST-p12融合蛋白进行多克隆抗体的制备及鉴定。方法:将构建好的融合表达载体pGEX-4T-1-DOC-1转化BL21(DE3)型大肠杆菌,用IPTG诱导表达,得到GST... 目的:为了进一步研究p12DOC-1基因及其蛋白在口腔癌发生中的作用与调控机制,将已表达纯化好的GST-p12融合蛋白进行多克隆抗体的制备及鉴定。方法:将构建好的融合表达载体pGEX-4T-1-DOC-1转化BL21(DE3)型大肠杆菌,用IPTG诱导表达,得到GST-p12融合蛋白,经亲和层析柱纯化后电泳,再割胶研磨作为抗原直接免疫家兔,经Western blot及ELISA检测抗体效价及特异性并做免疫组化分析。结果:得到较高纯度的GST-p12融合蛋白,获得了兔来源的抗p12DOC-1血清,经蛋白免疫印迹杂交反应及免疫组化分析证实所得的抗体具有较高的特异性和效价。结论:成功制备了兔抗人的p12DOC-1抗血清,为进一步研究p12DOC-1在口腔癌中发生缺失的机理打下基础。 展开更多
关键词 p12doc-1 蛋白 口腔癌缺失 融合蛋白 多克隆抗体 免疫组织化学
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人CDK2-AP1(DOC-1)酵母双杂交诱饵质粒自激活作用鉴定 被引量:2
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作者 倪前伟 孙沫逸 +5 位作者 戴建武 韩津 曹立雪 金伟 郑军 李建虎 《北京口腔医学》 CAS 2010年第1期5-7,17,共4页
目的验证诱饵质粒pBD-DOC-1在酵母双杂交系统的自激活性及毒性作用,为应用酵母双杂交系统(yeasttwo-hybrid system)筛选与p12DOC-1/CDK2-AP1相互作用的蛋白建立实验基础。方法将诱饵质粒pBD-DOC-1转化到酵母细胞MAV203中,检测诱饵蛋白... 目的验证诱饵质粒pBD-DOC-1在酵母双杂交系统的自激活性及毒性作用,为应用酵母双杂交系统(yeasttwo-hybrid system)筛选与p12DOC-1/CDK2-AP1相互作用的蛋白建立实验基础。方法将诱饵质粒pBD-DOC-1转化到酵母细胞MAV203中,检测诱饵蛋白有无毒性和自激活作用。同时利用对照质粒组筛选组氨酸(His)本底表达抑制剂3AT(3-氨基-1,2,4-三唑)的合适工作浓度。结果诱饵质粒pBD-DOC-1成功转化到酵母细胞MAV203中,对宿主酵母细胞无毒性,对报告基因无自激活作用。确定了组氨酸(His)本底表达抑制剂3AT(3-氨基-1,2,4-三唑)的合适工作浓度。结论诱饵质粒pBD-DOC-1可以用于酵母双杂交实验,为进一步运用酵母双杂交技术在人类组织cDNA文库中筛选与之相互作用的蛋白奠定了基础。 展开更多
关键词 p12doc-1/CDK2-Ap1 酵母双杂交 自激活
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