Lastick S M, Nielsen P J, McConkey E H
Mol Gen Genet. 1977 Apr 29;152(3):223-30. doi: 10.1007/BF00693074.
HeLa cell ribosomal protein S6, and the increase in its phosphorylation level that occurs after resuspending cells in fresh medium plus serum, were studied using two-dimensional gel electrophoresis. The maximum level of S6 phosphorylation occurs about 2 h after adding fresh medium and seum to cells that have been allowed to grow to high density; this results in an almost complete shift of the spot representing S6 in two-dimensional polacrylamide gels to a new location. Mixing experiments showed that the differences in the level of phosphorylation occur in vivo and are not an artifact of in vitro sample preparation. This method of stimulating S6 phosphorylation provides a convenient system for studying the functional significance of the phenomenon. Only one other ribosomal protein was detectably phosphorylated using [32P]-labeling and autoradiography of dried two-dimensional gels. The level of phosphorylation of this protein, L14, does not change after serum stimulation.
利用二维凝胶电泳研究了HeLa细胞核糖体蛋白S6，以及将细胞重悬于新鲜培养基加血清后其磷酸化水平的增加情况。在向已生长至高密度的细胞中添加新鲜培养基和血清后约2小时，S6磷酸化达到最高水平；这导致二维聚丙烯酰胺凝胶中代表S6的斑点几乎完全迁移到一个新位置。混合实验表明，磷酸化水平的差异发生在体内，并非体外样品制备的假象。这种刺激S6磷酸化的方法为研究该现象的功能意义提供了一个便利的系统。使用[32P]标记和干燥二维凝胶的放射自显影，仅检测到另一种核糖体蛋白发生了磷酸化。血清刺激后，这种蛋白L14的磷酸化水平没有变化。
