Nováková M, Dráberová E, Schürmann W, Czihak G, Viklický V, Dr-aber P
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic.
Cell Motil Cytoskeleton. 1996;33(1):38-51. doi: 10.1002/(SICI)1097-0169(1996)33:1<38::AID-CM5>3.0.CO;2-E.
Monoclonal antibodies were prepared against conserved synthetic peptide from the C-terminus of the gamma-tubulin and their specificity was confirmed by immunoblotting, competitive enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The antibodies decorated interphase centrosomes as well as half-spindles and midbodies in mitotic cells of various origin. The prepared antibodies were used to study the gamma-tubulin distribution in nocodazole and taxol-treated cells. In the cells recovering from the nocodazole treatment, gamma-tubulin was found in centers of all microtubule asters. Examination of relative location of gamma-tubulin and microtubule asters in taxol-treated mitotic cells 3T3, HeLa and PtK2 revealed that the number of taxol-induced microtubule asters exceeded the number of gamma-tubulin-positive spots. The gamma-tubulin was often found in the periphery of microtubule asters. Centrosomal phosphoprotein epitope detected by MPM-2 antibody colocalized with gamma-tubulin in taxol-treated mitotic cells. The presented data suggest that taxol-induced microtubule asters are in vivo nucleated independently of gamma-tubulin, and other minus-end nucleator(s) are necessary for formation of such asters. Alternatively, gamma-tubulin is present in subthreshold amounts undetectable by immunofluorescence.
制备了针对γ-微管蛋白C末端保守合成肽的单克隆抗体，并通过免疫印迹、竞争性酶联免疫吸附测定（ELISA）和免疫荧光确认了它们的特异性。这些抗体可标记不同来源有丝分裂细胞的间期中心体以及半纺锤体和中体。所制备的抗体用于研究诺考达唑和紫杉醇处理的细胞中γ-微管蛋白的分布。在从诺考达唑处理中恢复的细胞中，在所有微管星状体的中心发现了γ-微管蛋白。对紫杉醇处理的有丝分裂细胞3T3、HeLa和PtK2中γ-微管蛋白和微管星状体相对位置的检查显示，紫杉醇诱导的微管星状体数量超过γ-微管蛋白阳性斑点的数量。γ-微管蛋白经常出现在微管星状体的周边。MPM-2抗体检测到的中心体磷蛋白表位在紫杉醇处理的有丝分裂细胞中与γ-微管蛋白共定位。所呈现的数据表明，紫杉醇诱导的微管星状体在体内独立于γ-微管蛋白成核，并且其他负端成核剂对于此类星状体的形成是必需的。或者，γ-微管蛋白以免疫荧光检测不到的亚阈值量存在。
