Daduang S, Nagata S, Matsuda M, Yamori T, Onodera K, Fukui Y
Department of Applied Biological Chemistry, University of Tokyo, Japan.
Immunol Cell Biol. 1995 Apr;73(2):134-9. doi: 10.1038/icb.1995.21.
We have established two hybridomas producing mAb to the carboxyl terminal region of phosphatidylinositol-3 kinase 85 kDa subunit type alpha (p85 alpha). Analysis using deletion mutants of p85 revealed that epitopes for the two mAb were located on the border of the src homology 2 (SH2) sequence located at the carboxyl end of p85. They immunoprecipitated free p85 efficiently, but reactivity to p85 bound to p110 was very weak. Together with the mAb which we have reported previously, a panel of mAb that covered the various parts of p85 alpha was obtained. Using this panel, we characterized two mutants of p85 (70 and 50 kDa) expressed in the human colon carcinoma cell line, HCC2998. No wild-type p85 was detected in these cells. A mAb specific to the carboxyl terminal region detected p70 but not p50, suggesting that this region is missing in p50. The panel of mAb is a useful tool to use to analyse mutant forms of p85.
我们已经建立了两种杂交瘤，它们能产生针对磷脂酰肌醇-3激酶85 kDaα亚基（p85α）羧基末端区域的单克隆抗体（mAb）。使用p85的缺失突变体进行分析表明，这两种mAb的表位位于p85羧基末端的src同源2（SH2）序列边界。它们能有效地免疫沉淀游离的p85，但与结合到p110上的p85的反应性非常弱。连同我们之前报道的mAb，获得了一组覆盖p85α各个部分的mAb。使用该组mAb，我们对在人结肠癌细胞系HCC2998中表达的p85的两个突变体（70 kDa和50 kDa）进行了表征。在这些细胞中未检测到野生型p85。一种对羧基末端区域特异的mAb检测到了p70，但未检测到p50，这表明该区域在p50中缺失。该组mAb是用于分析p85突变形式的有用工具。
