JUMONJI DOMAIN INTERACTING PARTNERS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 我们正在研究两个Jumonji域包含蛋白质，JHD 2和ECM 5的相互作用结合伴侣。 使用市售蛋白A标记的菌株的第一IP未能导致任何相互作用的伴侣。 使用的酵母细胞的量增加至30 g/IP，因为两种蛋白质具有低拷贝/细胞数。 此外，还尝试了不同的IP缓冲液。 然而，执行伙伴并没有领导任何互动的伙伴。 随后，我们使用Rout实验室蛋白A标签标记了JHD 2和ECM 5。 ECM 5 IP现在导致SNT 2和RPD 3作为交互伙伴。 SNT 2和RPD 3依次用Rout lab蛋白A标签标记，并且相互IP证实了RPD 3 IP中SNT 2和ECM 5的存在以及SNT 2 IP中ECM 5和RPD 3的存在。 JHD 2 IP测试了3种不同的IP条件。 银染和蛋白质印迹数据证实了在所有3个IP中分离到JHD 2，但复合物的LC-MS分析仍有待进行。 计划将来对ECM 5和SNT 2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.