Hoffman J C, Mullins J M
Department of Biology, Catholic University of America, Washington, DC 20064.
Cell Motil Cytoskeleton. 1990;16(1):68-79. doi: 10.1002/cm.970160109.
Salt-extracted proteins of taxol-stabilized microtubules from Chinese hamster ovary cells arrested at mitosis were used to immunize mice for hybridoma production. From a group of related monoclonal antibodies (MAbs), one, C9, recognized an epitope on antigens localized by immunofluorescence microscopy to interphase centrosomes and nuclei. The availability of the nuclear antigen was cell cycle-dependent; however, permeabilization of cells before fixation revealed that the antigen was present throughout the cell cycle. The nuclear antigen was exposed during prophase and was released from the nucleus upon nuclear envelope breakdown filling the cytoplasm of the mitotic cell. Antigenic material re-accumulated at daughter nuclei and was concealed during G1 phase. Detergent extraction of the cytoplasmic antigen from mitotic cells enabled localization of antigens to centrosomes, kinetochores, and the furrowing region/midbody. Immunoblot analysis of cells of a variety of species of origin identified an approximate 250 kD polypeptide as corresponding to the nuclear antigen, whereas polypeptides of 107/117 kD as well as approximately 250 kD accounted for the mitotic cytoplasmic antigens. No polypeptides could be associated with antigens at centrosomes, kinetochores, or midbodies. This MAb joins the antibody preparations previously reported that describe nuclear antigens, or epitopes on antigens, enhanced at mitosis.
用从处于有丝分裂期的中国仓鼠卵巢细胞中提取的紫杉醇稳定微管蛋白免疫小鼠以制备杂交瘤。从一组相关的单克隆抗体（MAb）中，一种名为C9的抗体识别通过免疫荧光显微镜定位到间期中心体和细胞核的抗原上的一个表位。核抗原的可用性取决于细胞周期；然而，固定前对细胞进行通透处理显示该抗原在整个细胞周期中都存在。核抗原在前期暴露，并在核膜破裂时从细胞核中释放出来，充满有丝分裂细胞的细胞质。抗原物质在子细胞核处重新积累，并在G1期被隐藏。从有丝分裂细胞中用去污剂提取细胞质抗原可将抗原定位到中心体、动粒和沟区/中体。对多种来源物种的细胞进行免疫印迹分析，确定一种约250 kD的多肽对应于核抗原，而107/117 kD以及约250 kD的多肽构成有丝分裂细胞质抗原。没有多肽与中心体、动粒或中体上的抗原相关。这种单克隆抗体加入了先前报道的描述在有丝分裂期增强的核抗原或抗原上的表位的抗体制剂中。
