CUEDC2 Monoclonal Antibody,tet Inducible Regulated Expression Of Cell-free System Establishment And The Establishment Of Synchronized Methods

CUE domain containing protein 2(CUEDC2) is a ubiquitin-binding motif containing protein. CUE domain is a small conserved sequence containing approximately 40 amino acids, which can recognize both mono-ubiquitin and poly-ubiquitin. CUE domain containing proteins are widely involved in intracellular signaling responses. we found in our previous work that CUEDC2 interacts with progesterone receptor (PR) and inhibits progesterone signaling pathway by promoting progesterone-induced PR degradation through the ubiquitin-priteasome pathway. Moreover, we identify the sumoylation site Lys-388 of PR as the target of CUEDC2-promoted ubiquitination. CUEDC2 regulates PRB by decreasing the sumoylation while promoting ubiquitination on Lys-388. Our further study shows that CUEDC2 acts as an adaptor protein to target IKK for dephosphorylation and inactivation by recruiting GADD34 and a regulatory subunit of protein phosphatase 1(PP1), so as to repress activation of the NF-κB signaling pathway. Latest research reveals that CUEDC2 is a key factor for endocrine resistance in breast cancer. CUEDC2 modulates ER protein stability through the ubiquitin-proteasome pathway and leads to endocrine resistance. Our findings suggest CUEDC2 involving in multiple signaling pathways and playing an important role, though the functions are not yet clear.For the further study of CUEDC2, firstly we efforts to obtain CUEDC2 monoclonal antibodies of high purity and specificity. We immune mice with full-length CUEDC2 protein to get spleen cells producing CUEDC2 antibody, then fuse them with myeloma cells. through screening, identification and ascites collection, we eventually got CUEDC2 monoclonal antibodies, laying a foundation for the study of CUEDC2.A controlled gene expression system in cell lines and tissues serve as fundamental tools for function investigation, which accurately reflects the situation in vivo and provides reliable data. To get a stable cell line regulated by tet-off, we need to transfected tet-off plasmid and TER retrovirus recombinant plasmid into cells respectively, which usually indicates lots of workload and low efficiency. Today people attempt to build a mono-retroviral plasmid model to overcome the difficulties. Therefore, we choose p617-neo-T-EGFP-I-tTA4 vector, which make two systems up to one plasmid and get the non-toxic tTA4 instead of tTA, to construct CUEDC2 induced expression plasmid. Consequently the tet-off system gets a higher retrovirus titration and can be stricter on exogenous gene expression regulation.UV-induced DNA damage response is involved in multiple signaling pathways. When DNA is stimulated by certain factors (including physical damage, chemical stimulation, ultraviolet irradiation, ion irradiation, etc.), cells start a series of response like cell cycle arrest, DNA damage repair, cell senescence and apoptosis. CUEDC2 affects multiple signaling pathways and plays different roles, our previous study suggests CUEDC2 may involve in the regulation of cell cycle, so we established a UV-induced DNA damage model to study its function in cell cycle arrest from DNA damage, which may take great effort for subsequent research.