Analysis of G0 phase-specific large complex including p27

我们尝试通过分析方法鉴定GO特异性大复合物的组成，包括p27，以检测HEK 293 T细胞GO期培养的异步和生长停滞之间的差异。p27 - 1 <Ser 10A/Ser 10A>基因敲入小鼠脾脏和睾丸。与野生型细胞相比，p27^<Ser 10A/Ser 10A>小鼠淋巴细胞中G 0期p27的稳定性显着降低。然后，我们用凝胶过滤法对G 0期的p27进行了分析，发现p27在G 0期抑制剂中特异性地形成了大的复合物。通过FLAG抗体亲和层析和凝胶过滤层析，我们回收了一个游离的FLAG-p27，并收集了FLAG-p27相互作用蛋白。共鉴定出26个p27相互作用蛋白（包括13个新蛋白和13个已知蛋白）。然而，我们试图通过比较异步和生长停滞培养细胞之间的蛋白质来鉴定特异性地在G 0期与p27相关的蛋白质，我们未能成功。然而，我们发现PP 2A亚基A（Protein Phosphatase 2A，蛋白磷酸酶2A）作为p27的一种新的结合蛋白，对p27的稳定性变得不稳定起着重要作用。基于这一假设，我们正在使用磷酸酶抑制剂或针对PP 2A的siRNA来检查PP 2A对p27稳定性的影响。我们还计划使用NIH 3 T3鉴定p27的G 0特异性结合蛋白，就像正常细胞一样。

We have tried to identify the components of GO-specific large complex including p27 by analytical method for detection of difference between asynchronous and growth arrested in GO phase culture of HEK293T cells.Previously, we reported that the abundance of p27 was decreased in many organs, including brain, thymus. spleen and testis of p27^<Ser10A/Ser10A> knock-in mice. The stability of p27 in G0 phase was markedly reduced in lymphocytes of p27^<Ser10A/Ser10A> mice compared with wild-type cells. Then, we analyzed p27 in G0 phase by size fractionation using gel-filtration and obtained that p27 formed large complex specifically in G0 phase inhibitor. By affinity column chromatography using anti-FLAG antiboby and gel-filtration chromatography, we recovered a free-FLAG-p27, and could collect FLAG-p27 interacting proteins. Then we succeeded to identify 26 p27 interacting proteins (including 13 novel and 13 already-known) in total. Whereas we tried to identify the proteins were associated to p27 specifically in G0 phase by comparison of proteins between asynchronous and growth arrested culture cells, we could not succeed. However, we identified PP2A subunit A (Protein Phosphatase 2A) as a novel binding protein of p27 plays an important role for the stability of p27 becomes to be unstable. Based on this hypothesis, we are examining the effects of PP2A on stability of p27 using phosphatase inhibitor or siRNA against PP2A. We also plan to identify the G0-specific binding proteins of p27 using NIH 3T3, like as normal cells.