JUMONJI DOMAIN INTERACTING PARTNERS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 我们正在研究两个含有Jumonji结构域的蛋白JHD2和ECM5的相互作用结合伙伴。使用商业上可获得的A蛋白标记菌株的第一批IP未能导致任何相互作用的伙伴。酵母细胞的使用量增加到每个IP 30g，因为这两种蛋白质的拷贝/细胞数量都很低。此外，还尝试了不同的IP缓冲区。然而，国际合作伙伴并没有领导任何互动伙伴。随后，我们使用路由器实验室蛋白A标签自己标记了JHD2和ECM5。ECM5 IP现在导致SNT2和RPD3成为交互伙伴。SNT2和RPD3依次被标记有路由器实验室蛋白A标签，并且相互的IP证实在RPD3 IP中存在SNT2和ECM5，在SNT2 IP中存在ECM5和RPD3。对JHD2 IP进行了3种不同的IP测试。银染和Western印迹数据证实在所有3个IP中都分离到JHD2，但对该复合体的LC-MS分析仍有待进行。未来计划对ECM5和SNT2进行PTM分析。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We are studying the interacting binding partners of two Jumonji domain containing proteins, JHD2 and ECM5. The first IPs using commercially available protein A tagged strains failed to lead to any interacting partners. The amount of yeast cells used was increased to 30 g per IP, as both proteins have low copy/cell numbers. In addition, different IP buffers were tried. Nevertheless, the IPs did not lead any interacting partners. Subsequently, we tagged JHD2 and ECM5 ourselves using the Rout lab protein A tag. ECM5 IP now resulted in SNT2 and RPD3 as interacting partners. SNT2 and RPD3 were in turn tagged with the Rout lab protein A tag and the reciprocal IPs confirmed the presence of SNT2 and ECM5 in the RPD3 IP and the presence of ECM5 and RPD3 in the SNT2 IP. The JHD2 IP was carried out testing 3 different IP conditions. Silver stain and Western blot data confirmed isolation of JHD2 in all 3 IPs, but the LC-MS analysis of the complex is still pending. PTM analysis of ECM5 and SNT2 are planned for the future.