Hart C E, Nuckolls G H, Wood J G
Cell Motil Cytoskeleton. 1987;7(4):393-403. doi: 10.1002/cm.970070411.
Immunocytochemistry and polyacrylamide gel electrophoresis have been used to study the distribution of phosphorylated forms of neurofilament antigens in rat brain. Immunostaining of tissue with an antisera produced against phosphatase-sensitive domains of the 200-kilodalton (kd) neurofilament polypeptide showed that phosphorylated forms of this polypeptide were present in virtually all axons and certain somata and dendrites of neurons in different brain regions. Immunoblots of whole brain homogenate or a neurofilament preparation from rat revealed that the affinity-purified anti-200-kd sera used to immunostain tissue labeled the neurofilament-associated 200-kd band in a phosphatase-sensitive manner. Fine structural analysis of this immunoreactivity in tissue showed that whenever the labeled organelle could be identified, it was a microtubule. In contrast, immunoblot analysis of twice-cycled microtubules from porcine brain revealed that microtubules in vitro did not possess the 200-kd antigen that was observed in situ. The results suggest that our antibody recognizes a phosphorylated domain on the neurofilament involved in cross-linking neurofilaments and microtubules, and that in vivo, phosphorylated epitopes of the 200-kd neurofilament polypeptide are capable of associating with microtubules.
免疫细胞化学和聚丙烯酰胺凝胶电泳已被用于研究大鼠脑中神经丝抗原磷酸化形式的分布。用针对200千道尔顿（kd）神经丝多肽磷酸酶敏感结构域产生的抗血清对组织进行免疫染色，结果表明该多肽的磷酸化形式几乎存在于不同脑区神经元的所有轴突以及某些胞体和树突中。对大鼠全脑匀浆或神经丝制剂进行免疫印迹分析发现，用于组织免疫染色的亲和纯化抗200-kd血清以磷酸酶敏感的方式标记了神经丝相关的200-kd条带。对组织中这种免疫反应性的精细结构分析表明，只要能够识别出标记的细胞器，它就是微管。相比之下，对猪脑经两次循环的微管进行免疫印迹分析发现，体外微管不具有原位观察到的200-kd抗原。结果表明，我们的抗体识别神经丝上参与神经丝与微管交联的磷酸化结构域，并且在体内，200-kd神经丝多肽的磷酸化表位能够与微管结合。
