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    Appl proteins as rab5 effectors
    6.
    发明申请
    Appl proteins as rab5 effectors 审中-公开
    应用蛋白作为rab5效应物

    公开(公告)号：US20070020696A1

    公开(公告)日：2007-01-25

    申请号：US10564435

    申请日：2004-07-08

    申请人： Marino Zerial ,  Marta Miaczynska

    发明人： Marino Zerial ,  Marta Miaczynska

    IPC分类号： G01N33/567 ,  G01N33/53

    CPC分类号： C07K14/4702

    摘要： The present invention relates to an in vivo-assay to screen for anti-proliferative drugs, the assay comprising the steps of: (a) contacting cells of a primary cell culture or of an established cell line with a candidate substance, (b) subsequently or concomitantly with a candidate substance, contacting the cells with a growth factor, (c) processing the cells for immunofluorescence staining to detect APPL1 and APPL2 using an anti-APPL1 and/or 2 antibody, or alternatively using GFP-tagged APPL proteins stably or transiently expressed by the cells via transfection, (d) assessing the degree of colocalisation of APPL1 and/or 2 and the growth factor, the solubilisation of APPL1 and/or 2 and their translocation to the nucleus, (e) repeating steps (b) to (d) with cells not previously treated with the candidate substance, and (f) comparing the degree of colocalisation of APPL1 and/or 2 and the growth factor, the solubilisation of APPL1 and/or 2 and their translocation to the nucleus between the cells not previously treated with the candidate substance (untreated cells) and cells treated with the candidate substance (treated cells), wherein an altered degree of colocalisation of APPL1 and/or 2 and the growth factor, an altered solubilisation of APPL1 and/or 2 and/or their altered translocation to the nucleus in the treated vs. the untreated cells identifies the candidate substance as an anti-proliferative drug.

    摘要翻译： 本发明涉及筛选抗增殖药物的体内测定法，该测定法包括以下步骤：（a）将原代细胞培养物或已建立的细胞系的细胞与候选物质接触，（b）随后 或伴随着候选物质，使细胞与生长因子接触，（c）使用抗-PL1和/或2抗体处理细胞进行免疫荧光染色以检测APPL1和APPL2，或者使用GFP标记的APPL蛋白稳定地或 （d）评估APPL1和/或2的共定位程度和生长因子，APPL1和/或2的溶解及其向细胞核的易位，（e）重复步骤（b） 至（d）未预先用候选物质处理的细胞，和（f）比较APPL1和/或2与生长因子的共定位程度，APPL1和/或2的溶解以及它们在细胞核之间的易位 先前未用候选物质（未处理的细胞）处理的细胞和用候选物质处理的细胞处理的细胞（经处理的细胞），其中APPL1和/或2和生长因子的共定位程度改变APPL1和/或2的溶解度改变 和/或其改变的处理相对于未处理细胞中的细胞核的易位将候选物质鉴定为抗增殖药物。