Senden N H, Timmer E D, Boers J E, van de Velde H J, Roebroek A J, Van de Ven W J, Broers J L, Ramaekers F C
Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, Netherlands.
Eur J Cell Biol. 1996 Mar;69(3):197-213.
A mouse monoclonal antibody RNL-4, as well as rabbit polyclonal antiserum POL-8 were raised against a synthetic peptide, encompassing the first twenty unique amino-terminal amino acid residues of NSP-C. The specificity of both immunoreagents was established in an ELISA assay using the synthetic peptide and by their immunoreactivity to NSP-C fusion proteins. Immunofluorescence analysis of COS-1 cells, transfected with NSP-C cDNA, showed staining of the endoplasmic reticulum with RNL-4 and POL-8. No cross-reactivity of these reagents with NSP-A or NSP-B was seen. Immunohistochemical studies in normal human tissues showed expression of NSP-C in tissues of neural and neuroendocrine origin, i.e. neurons of the central and peripheral nervous system, the neurohypophysis, adrenal medulla, adenohypophysis, pars intermedia, and in sporadic neuroedocrine cells of the lung. Expression of NSP-C was found in several small cell lung cancer (SCLC) cell lines, in non-SCLC cell lines with neuroendocrine features, but not in typical non-SCLC cell lines. Also, in a neuroblastoma cell line NSP-C expression was observed. Immunoblotting and immunoprecipitation studies with RNL-4 and POL-8 identified the 23 kDa NSP-C polypeptide in these cell lines. Immunofluorescence microscopy showed that also in these cell lines NSP-C is located at the endoplasmic reticulum, as shown before for NSP-A and NSP-B. In some of the cell lines coexpression of NSP-A and NSP-C was observed, while in others only one of the two could be detected. The differential expression of NSP-A and NSP-C in these cell lines is confirmed by immunoblotting and was also evident at the mRNA level. When NSP-A and NSP-C were coexpressed, the number of NSP-C-positive cells was always less than the number of NSP-A-positive cells. A partial colocalization of NSPs was observed in the endoplasmic reticulum. Cell fractionation studies revealed that both proteins are retained in the membranous fraction of the cell, from which they can be solubilized by Triton X-100. Immunoprecipitation analyses under native conditions indicate that NSP-C does not need to associate with NSP-A to form high molecular weight NSP-reticulons.
针对包含NSP-C前20个独特氨基末端氨基酸残基的合成肽，制备了小鼠单克隆抗体RNL-4以及兔多克隆抗血清POL-8。通过使用合成肽的ELISA测定以及它们与NSP-C融合蛋白的免疫反应性，确定了这两种免疫试剂的特异性。对用NSP-C cDNA转染的COS-1细胞进行免疫荧光分析，结果显示RNL-4和POL-8对内质网有染色。未观察到这些试剂与NSP-A或NSP-B有交叉反应。在正常人体组织中的免疫组织化学研究表明，NSP-C在神经和神经内分泌起源的组织中表达，即中枢和外周神经系统的神经元、神经垂体、肾上腺髓质、腺垂体、中间部，以及肺的散在神经内分泌细胞。在几种小细胞肺癌（SCLC）细胞系、具有神经内分泌特征的非SCLC细胞系中发现了NSP-C的表达，但在典型的非SCLC细胞系中未发现。此外，在一个神经母细胞瘤细胞系中也观察到了NSP-C的表达。用RNL-4和POL-8进行的免疫印迹和免疫沉淀研究在这些细胞系中鉴定出了23 kDa的NSP-C多肽。免疫荧光显微镜显示，在这些细胞系中NSP-C也位于内质网，这与之前对NSP-A和NSP-B的观察结果一致。在一些细胞系中观察到了NSP-A和NSP-C的共表达，而在其他细胞系中只能检测到两者之一。通过免疫印迹证实了这些细胞系中NSP-A和NSP-C的差异表达，在mRNA水平上也很明显。当NSP-A和NSP-C共表达时，NSP-C阳性细胞的数量总是少于NSP-A阳性细胞的数量。在内质网中观察到了NSPs的部分共定位。细胞分级分离研究表明，这两种蛋白质都保留在细胞的膜部分，可通过Triton X-100将它们溶解。在天然条件下的免疫沉淀分析表明，NSP-C不需要与NSP-A结合就能形成高分子量的NSP-网蛋白。
