The Functional Study Of PBDC1 And NonO In Erythroid Differentiation And Antibody Preparation Of UPF0538

Cell differentiation and dedifferentiation is a research focus in the life science, the investigation of its mechanism can provide some important information dates for tumor and its therapy researches. Erythroid differentiation of mammals has scheduling changes, such as cell division stopping, cell volume decrease, nuclear pyknosis denucleation and active expression of hemoglobin gene. Murine fetal liver is an important early-statage blood-forming organ, erythroblast occur and finish, hematoiesis in E10.5-E17.5 fetal liver, so fetal liver is a good model for the study of cell differentiation. Morevevr, chemicals like sodium butyrate, dimethylsulfoxide and hexamethylene bisacetamide can induce erythroleukemia cells to regain its differentiation capacity and undergo normal differentiation.In the early stage of the work carried out in our laboratory, we had taken use of comparative proteomics technology to identify many differentially expressed proteins during Murine erythroleukemia cell induced differentiation by sodium butyrate. Part of Which were some proteins with a certain basis function research, such as, NPM1, Pirin, RbAp48, NonO, and others were some unknown proteins, such as, PBDC1, UPF0538.We studied the function of protein PBDC1 and NonO during erythroid differentiation by celluar and molecular methods, and prepared polyclonal antibody of protein UPF0538 in this paper as well. PBDC1 protein polyclonal antibody and its expression at gene and protein level during MEL induced differentiation by sodium butyrate were made previously. In this paper, our study found that PBDC1 mainly located in cytoplasm in MEL cells and mouse fetal liver cells located in cytoplasm, and a small amount in nuclei. A small part of PBDC1 protein would be eliminated accompanied by natural enucleation, mainly of which would stay in enucleated red blood cells.This probably suggested that PBDC1 protein may be associated with certain features of mature red blood cells.159 proteins were identified as interacted with PBDC1. Among them, enzymes,especially peptidase and oxidoreductase accounted for a large proportion, others are GTP binding proteins, proteins with electron carrier ability, iron binding proteins and so on. The results indicated that PBDC1 was probably involved in intracellular process such as oxidoreduction reaction, electron transfer, iron binding and so on.Previously, we found that in the process of MEL induced differentiation by sodium butyrate, NonO protein expression was increased, and co-located with erythroid deregulation factor PU.1, suggesting that NonO may participate in erythroid differentiation regulation. In this paper, we conducted stable cell lines by knockdown of NonO protein, and its MTT assay indicated inhibition of cell viability since downregulation of NonO. Moreover, low expression of NonO caused decline of a significant erythroid differentiation regulator c-myc. Above all, it was suggested that NonO regulated erythroid differentiation through c myc and PU.1.UPF0538 was a unknown function protein and there was no commercial antibodies. In order to study its function on erythroid, we chose prokaryotic expression system to acquire plenty of UPF0538 fusion protein so that we could get UPF0538 polyclonal antibody by immuning the New Zealand white rabbit, thus providing useful material for its function research.