The methyl-esterification of the C-terminal leucine of the protein phosphatase 2A (PP2A) catalytic (C) subunit is essential for the assembly of specific trimeric PP2A holoenzymes, and this region of the C subunit also contains two threonine and tyrosine phosphorylation sites. Most commercial antibodies-including the monoclonal antibody 1D6 that is part of a frequently used, commercial phosphatase assay kit-are directed toward the C terminus of the C subunit, raising questions as to their ability to recognize methylated and phosphorylated forms of the enzyme. Here, we tested several PP2A C antibodies, including monoclonal antibodies 1D6, 7A6, G-4, and 52F8 and the polyclonal antibody 2038 for their ability to specifically detect PP2A in its various modified forms, as well as to coprecipitate regulatory subunits. The tested antibodies preferentially recognized the nonmethylated form of the enzyme, and they did not coimmunoprecipitate trimeric holoenzymes containing the regulatory subunits B or B', an issue that precludes their use to monitor PP2A holoenzyme activity. Furthermore, some of the antibodies also recognized the phosphatase PP4, demonstrating a lack of specificity for PP2A. Together, these findings suggest that reinterpretation of the data generated by using these reagents is required.

中文翻译：

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识别PP2A催化亚基C末端的抗体不适用于评估PP2A活性和全酶组成。

蛋白质磷酸酶2A（PP2A）催化（C）亚基C末端亮氨酸的甲基化对于特定三聚PP2A全酶的组装至关重要，并且C亚基的该区域还包含两个苏氨酸和酪氨酸磷酸化位点。大多数市售抗体（包括作为常用的市售磷酸酶测定试剂盒的一部分的单克隆抗体1D6）都指向C亚基的C末端，这引发了有关它们识别酶的甲基化和磷酸化形式的能力的疑问。在这里，我们测试了几种PP2A C抗体，包括单克隆抗体1D6、7A6，G-4和52F8以及多克隆抗体2038，它们能够特异性检测其各种修饰形式的PP2A并共沉淀调节亚基。被测抗体优先识别该酶的非甲基化形式，并且它们不能共免疫沉淀含有调节亚基B或B'的三聚全酶，这一问题使它们无法监测PP2A全酶的活性。此外，某些抗体也识别磷酸酶PP4，这表明对PP2A缺乏特异性。总之，这些发现表明需要重新解释使用这些试剂产生的数据。