Yen S H, Fields K L
J Cell Biol. 1981 Jan;88(1):115-26. doi: 10.1083/jcb.88.1.115.
Antisera were raised to the 210,000-dalton and the 49,000-dalton proteins of a fraction enriched in intermediate (10 nm) filaments from human brain. Proteins of the filament preparation were separated by SDS-polyacrylamide gel electrophoresis and used for immunization and subsequent analysis of the reactions of the sera by rocket immunoelectrophoresis. Anti-210,000-dalton serum precipitated proteins of molecular weights 210,000, 160,000, and 68,000, and, thus, reacted with all the neurofilament triplet components. Anti-49,000-dalton serum did not react with the triplet proteins but precipitated the 49,000-dalton protein. By immunofluorescence on tissue sections, anti-210,000-dalton serum bound to neuronal axons in sciatic nerve and cerebellum. In dissociated cell cultures, rat dorsal root ganglion cells and their processes bound the serum, whereas nonneuronal cells did not. Some cultured cerebellar neurons were also positive, whereas astrocytes were not. At the ultrastructural level, anti-210,000-dalton serum bound to intermediate filaments inside axonal processes. Anti-49,000-dalton serum bound to astrocytes in sections of the cerebellum, and cultured astrocytes had filaments that stained, whereas other cell types did not. In sciatic nerve sections, elements stained with this serum, but cultured cells from newborn sciatic nerve were negative. An antiserum against the 58,000-dalton protein of the cytoskeleton of NIL-8 fibroblasts strongly stained sciatic nerve sections, binding to Schwann cells but not to axons or to myelin. In cerebellar sections, astrocytes were positive, as were blood vessels and cells in the pia. In cell cultures, anti-58,000-dalton serum stained filaments inside Schwann cells, fibroblasts, and astrocytes, but neurons were negative. Cells in the cultures and tissue sections of the nervous system failed to react with antiserum to the 58,000-dalton protein of skin intermediate filaments. In these studies, astrocytes in vivo and in culture were the only cells which had antigens related to two classes of intermediate filaments.
制备了针对人脑中富含中间丝（10纳米）的组分中210,000道尔顿和49,000道尔顿蛋白质的抗血清。将丝体制备物中的蛋白质通过SDS-聚丙烯酰胺凝胶电泳分离，用于免疫，并随后通过火箭免疫电泳分析血清反应。抗210,000道尔顿血清沉淀出分子量为210,000、160,000和68,000的蛋白质，因此与所有神经丝三联体成分发生反应。抗49,000道尔顿血清不与三联体蛋白质反应，但沉淀出49,000道尔顿的蛋白质。通过对组织切片进行免疫荧光检测，抗210,000道尔顿血清与坐骨神经和小脑中的神经元轴突结合。在解离细胞培养物中，大鼠背根神经节细胞及其突起与该血清结合，而非神经细胞则不结合。一些培养的小脑神经元也呈阳性，而星形胶质细胞则为阴性。在超微结构水平上，抗210,000道尔顿血清与轴突内的中间丝结合。抗49,000道尔顿血清与小脑中的星形胶质细胞结合，培养的星形胶质细胞中有染色的丝，而其他细胞类型则没有。在坐骨神经切片中，该血清染色的成分，但新生坐骨神经的培养细胞为阴性。针对NIL-8成纤维细胞细胞骨架中58,000道尔顿蛋白质的抗血清强烈染色坐骨神经切片，与施万细胞结合，但不与轴突或髓鞘结合。在小脑切片中，星形胶质细胞呈阳性，血管和软膜中的细胞也呈阳性。在细胞培养物中，抗58,000道尔顿血清对施万细胞、成纤维细胞和星形胶质细胞内的丝进行染色，但神经元为阴性。神经系统的培养细胞和组织切片不与针对皮肤中间丝58,000道尔顿蛋白质的抗血清发生反应。在这些研究中，体内和培养中的星形胶质细胞是唯一具有与两类中间丝相关抗原的细胞。
