摘要
通过制备、鉴定与筛选可靠应用于反相蛋白阵列(reverse-phase protein array,RPPA)的磷酸化抗体作为探针,建立LNCa P细胞中Akt、Erk/MAPK通路在4种方式(EGF、LY294、PMA、Na Cl)刺激下信号变化的细胞通路模型,利用RPPA技术探测细胞通路模型的信号。结果获得4组细胞通路模型的RPPA测定数据及25个RPPA适用性的磷酸化抗体探针。4组模型结果均显示了通路中关键信号分子响应于4个外部扰动的磷酸化状态的动态变化。RPPA结果数据与相应的免疫印迹法(Western blot)分析结果一致,证实了分析数据的有效性与可靠性。结果表明,所制备磷酸化抗体探针与RPPA平台结合,极适合于分析细胞信号通路模型。该方法能够有效地拓展RPPA探针范围,使其成为新药开发、临床样本分析等领域的先进工具。
Phospho-specific antibodies as probes with reliability for application in reverse-phase protein array（RPPA） were prepared, characterized and screened. Combined with RPPA, they were used to explore signals of cell signaling pathway patterns. The cell signaling pathway patterns were established using Akt, Erk/MAPK pathways in LNCa P cells which generate signal changes under 4 treatment methods（EGF, LY294, PMA, Na Cl）. 4 groups of patterns data by RPPA assay were ultimately obtained, as well as 25 phospho-specific antibodies probes with high availability for RPPA. 4 groups data all showed dynamic changes of the phosphorylation status of key signaling moleculars in the pathways which responded for cell external treatments. The RPPA assay data were consistent with the results of Western blot which approved the validity and reliability of these data. The results showed that these phospho-specific antibody probes combined with the RPPA platform were very suitable for the analysis of cell signaling pathway patterns. This method can effectively expand the RPPA antibody probes, and make it an advanced tool in the fields of new drug discovery, clinical samples analysis etc.
作者
林世康
孙钵清
王琪炜
胡云龙
顾宁
Lin Shikang;Sun Boqing;Wang Qiwei;Hu Yunyong;Gu Ning(School of Biological Science and Medical Engineering,Southeast University,Nanjing 210096,China;Nanjing Chuanbo Biotech.Co.,Ltd.,Nanjing 210031,China)
出处
《中国细胞生物学学报》
CAS
CSCD
2018年第7期1193-1201,共9页
Chinese Journal of Cell Biology
关键词
磷酸化抗体
细胞信号转导
通路
反相蛋白阵列
phospho-specific antibody
cell signal transduction
pathway
reverse-phase protein array
