Giardina S L, Storm S M, Longo D L, Mathieson B J, Rapp U, Varesio L
Program Resources, Inc., National Cancer Institute-Frederick Cancer Research Facility, MD 21701.
J Immunol. 1988 May 15;140(10):3528-33.
A murine mAb, STEGI 1, was generated against a 30-kDa raf protein purified from an Escherichia coli expression vector. Immunoblot analysis confirmed that this antibody recognized the original immunizing protein as well as a 44- to 48-kDa protein from several raf-transformed cell lines. Immunoprecipitation experiments isolated a 48-kDa protein from a cell line transfected with a c-raf construct as well as from normal NIH 3T3 fibroblasts. Parallel experiments with polyvalent antiserum prepared against E. coli-derived v-raf (C terminus)-precipitated proteins with apparent Mr of 48 and 74 kDa, as had been described previously. Immunofluorescence flow cytometry of raf-transformed cell lines revealed intense intracytoplasmic staining. This staining was specifically inhibited by preincubation of STEGI 1 with purified raf 30-kDa protein. It should now be possible to more easily assess the role of the raf oncogene product in malignant transformation.
针对从大肠杆菌表达载体中纯化的30 kDa raf蛋白制备了一种鼠单克隆抗体STEGI 1。免疫印迹分析证实，该抗体可识别原始免疫蛋白以及来自几种raf转化细胞系的44至48 kDa蛋白。免疫沉淀实验从转染了c-raf构建体的细胞系以及正常NIH 3T3成纤维细胞中分离出一种48 kDa蛋白。与针对大肠杆菌衍生的v-raf（C末端）制备的多价抗血清进行的平行实验沉淀出了表观分子量为48和74 kDa的蛋白，如先前所述。raf转化细胞系的免疫荧光流式细胞术显示强烈的胞质内染色。用纯化的30 kDa raf蛋白预孵育STEGI 1可特异性抑制这种染色。现在应该能够更轻松地评估raf癌基因产物在恶性转化中的作用。
