Dziadek M, Timpl R
Dev Biol. 1985 Oct;111(2):372-82. doi: 10.1016/0012-1606(85)90491-9.
Nidogen and laminin were localized at preimplantation stages of mouse development by immunofluorescence. Laminin was already present on the cell surface at the 2-cell stage, while nidogen was first detectable on compacted 8- to 16-cell stage morulae. Nidogen and laminin colocalized at the blastocyst stage and in postimplantation basement membranes. Immunoblot analyses of tissue extracts and cell culture media indicated the 150-kDa form of nidogen as the largest and predominant form in all tissues examined. Radiolabeled nidogen and laminin synthesized by Reichert's membrane were coprecipitated by antibodies against each antigen, indicating complex formation in situ. Equimolar amounts of laminin and nidogen were determined in 6 M guanidine X HCl extracts of tissues by radioimmunoassays, further indicating stoichiometric complexes. However, lower levels of nidogen than laminin were found in tissue and cell culture media. A less than 2-fold increase in nidogen was found when F9 cells were stimulated to differentiate with retinoic acid and dibutyryl cAMP, compared to a 30-fold increase in laminin secretion.
通过免疫荧光法确定了巢蛋白和层粘连蛋白在小鼠发育植入前阶段的定位。层粘连蛋白在2细胞阶段就已出现在细胞表面,而巢蛋白最早在紧密化的8至16细胞期桑椹胚中可检测到。巢蛋白和层粘连蛋白在囊胚期和植入后基底膜中共定位。对组织提取物和细胞培养基的免疫印迹分析表明,150 kDa形式的巢蛋白是所有检测组织中最大且最主要的形式。由Reichert膜合成的放射性标记的巢蛋白和层粘连蛋白可被针对每种抗原的抗体共沉淀,表明原位形成复合物。通过放射免疫测定法在组织的6 M盐酸胍提取物中测定了等摩尔量的层粘连蛋白和巢蛋白,进一步表明了化学计量复合物的存在。然而,在组织和细胞培养基中发现巢蛋白的水平低于层粘连蛋白。与层粘连蛋白分泌增加30倍相比,当用视黄酸和二丁酰环磷腺苷刺激F9细胞分化时,巢蛋白的增加不到2倍。