This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.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.
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