Wiche G, Briones E, Koszka C, Artlieb U, Krepler R
EMBO J. 1984 May;3(5):991-8. doi: 10.1002/j.1460-2075.1984.tb01918.x.
The occurrence and cellular localization of polypeptides related to hog brain microtubule-associated proteins 1 and 2 (MAP-1 and MAP-2) in non-neuronal cell lines of various species and types, and in several tissues from rat was studied. When insoluble cell fractions were prepared by incubation of isotonic cell extracts with 20 microM taxol, polypeptides co-migrating with MAP-1 and MAP-2 upon gel electrophoresis were observed in virtually all cases examined. Immunoblotting of preparations from 3T6, CHO, HeLa and N2A cells, as well as pituitary, heart, testis and liver revealed immuno-reactivity with antibodies to neuronal MAP-1 for polypeptides co-migrating with MAP-1 in all cases, except for HeLa cells and liver. With similar preparations, antibodies raised to neuronal MAP-2 were barely reactive with bands of the MAP-2 size except for N2A cells and pituitary gland. In all cases of non-neuronal cells and tissues, major cross-reactive bands, however, were of mol. wt. lower than that of MAP-2, indicating, most likely, proteolytic breakdown of MAP-2 during cell fractionation. As shown by double immunofluorescence microscopy of various cultured cell lines using affinity-purified antibodies to MAPs, and monoclonal antibodies to tubulin, MAP-1-as well as MAP-2-related antigens were generally, but not exclusively, associated with typical microtubule structures of the cytoplasm, spindle, midbody and primary cilia. Antigens related to both MAPs were also localized in frozen sections of rat trachea, testis, pituitary, kidney and cardiac and skeletal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)
研究了与猪脑微管相关蛋白1和2(MAP-1和MAP-2)相关的多肽在各种物种和类型的非神经元细胞系以及大鼠的几种组织中的发生情况和细胞定位。当通过将等渗细胞提取物与20微摩尔紫杉醇孵育来制备不溶性细胞组分时,在几乎所有检测的情况下,在凝胶电泳中观察到与MAP-1和MAP-2共迁移的多肽。对3T6、CHO、HeLa和N2A细胞以及垂体、心脏、睾丸和肝脏的制剂进行免疫印迹分析,结果显示,除HeLa细胞和肝脏外,在所有情况下与MAP-1共迁移的多肽都与抗神经元MAP-1抗体发生免疫反应。对于类似的制剂,除了N2A细胞和垂体腺外,针对神经元MAP-2产生的抗体与MAP-2大小的条带几乎没有反应。然而,在所有非神经元细胞和组织的情况下,主要的交叉反应条带的分子量低于MAP-2,这很可能表明在细胞分级分离过程中MAP-2发生了蛋白水解降解。使用针对MAPs的亲和纯化抗体和针对微管蛋白的单克隆抗体对各种培养细胞系进行双重免疫荧光显微镜检查,结果表明,MAP-1以及与MAP-2相关的抗原通常但并非仅与细胞质、纺锤体、中体和初级纤毛的典型微管结构相关。与这两种MAPs相关的抗原也定位在大鼠气管、睾丸、垂体、肾脏以及心脏和骨骼肌的冰冻切片中。(摘要截断于250字)