Functional Study Of Several Hydrolyzed Fragments From Retinoblastoma Protein/Study On The Interaction Of Sedlin With DEPP

Functional Study of Several Hydrolyzed Fragments from Retinoblastoma ProteinObjective The main purpose of this topic is to study the function s of several h ydrolyzed fragments of R b in cells.The eukaryotic expression plasmid of truncated forms of the Rb1 gene were constructed,and transfected into mammalian cells to allow the cellu lar localization of Rb truncated forms to be observe.In the meantime,the role in cell cycle progression and apoptosis were detected by overexpression of those truncated forms.Methods The eukaryotic expression plasmids of pc DNA3.1-p76^{R b}-FLAG,pc DNA3.1-p80^{R b}-FLAG and pc DNA3.1-p100^{R b}-FLAG were constructed with pc DNA3.1-RB1-FLAG as template.U2OS cells were transfected with those plasmids respectively.After U2OS cells were grown on a slide for 24hours,cells on the slide were treated by immunofluorescence experiment to observe the localization of truncated forms of Rb.HEK 293T cells were transfected with truncated forms of Rb respectively.48 hours after transfection,the cells were collected and lysed for Western blotting.The pc DNA3.1-p76^{R b}-FLAG was transiently transfected into HEK 293T cells and 20μM MG132 was added 8 hours before receiving cells.The protein extracted from the cells was collected and Western blotting was performed.The pc DNA3.1-p80^{R b}-FLAG,pc DNA3.1-p100^{R b}-FLAG and pc DNA3.1-RB1-FLAG were transiently transfected into HEK 293T cells respectively.24hours after transfection,the cells we re collected to detect the changes of cell cycle and apoptosis by flow cytometry.The pc DNA3.1-RB1-FLAG and pc DNA3.1-RB1-HA were transiently transfected into HEK 293T cells.48hours after transfection,proteins were collected and immunoprecipitation was performed.Results Enzyme digestion and sequencing analysis demonstrated those plasmids of pc DNA3.1-p76^Rb-FLAG、pc DNA3.1-p80^Rb-FLAG、pc DNA3.1-p100^Rb-FLAG were correctly constructed.The results of immunofluorescence showed that p76^Rb and p100^Rb were mainly located in the nucleus of U2OS cells,p80^Rbwas mainly located in the cytoplasm in U2OS cells.The results of WB showed that p80^{R b} and p100^{R b} were overexpressed,and p76^Rb could be overexpressed after MG132 proteasome inhibitor was added.The results of cell cycle and apoptosis detected by flow cytometry showed that the percentage of G1 phase of cell cycle in overexpressed Rb experimental group and overexpressed p100^{R b} experimental group was higher than that in control group,and the apoptosis rate in each experimental group was significantly lower than that in control group.Furthermore,the results of immunoprecipitation showed that Rb may exist in dimer form.Conclusion The p80^{R b} and p100^{R b} can be expressed in HEK 293T cells,but p76^{R b} can be degraded through proteasome pathway,suggesting that the N-terminal domain of Rb protein can stabilize Rb protein in HEK 293T cells;Overexpression of p100^{R b} and Rb can block cell cycle;Overexpressed p80^{R b},p100^{R b} and Rb can inhibit apoptosis,suggesting that the N-terminal domain or P-terminal domain of Rb protein is related to the apoptosis inhibition of Rb in HEK 293T cells.Study on the Interaction of Sedlin with DEPPObjective the main purpose of this topic is to study the interaction between DEPP and its deletion mutants and Sedlin.Western blotting,GST pulldown and exogenous immunoprecipitation were used to explore the expression of DEPP deletion mutants and confirm the specific domains of the interaction between DEPP and Sedlin.Methods HEK 293 T cells were transfected with pc DNA3.1-DEPP-FLAG、pc DNA3.1-DEPP（1-100aa）-N-FLAG 、 pc DNA3.1-DEPP（101-112aa）-C-FLAG 、pc DNA3.1-DEPP（ΔPPPSP）-FLAG 、 pc DNA3.1-DEPP（S34A）-FLAG respectively.48 hours after of transfection,the cells were collected and lysed for Western blotting.HEK 293 T cells were transfected with pc DNA3.1-DEPP-FLAG、pc DNA3.1-DEPP-N-FLAG 、 pc DNA3.1-DEPP（ΔPPPSP）-FLAG 、 pc DNA3.1-DEPP（S34A）-FLAG respectively.After transfection for 48 hours,the c ells were collected to extract total protein,which was mixed with purified GST Sedlin fusion protein and incubated in vitro.After incubation the Glutathione beads were collected,then the samples were prepared for Western blotting.pc DNA3.1-DEPP-FLAG and pc DNA3.1-Sedlin-HA were transiently transfected into HEK 293 T cells.After 48 hours of transfection,the total protein was extracted and treated with FLAG antibody for Co-immunoprecipitation.Results GST-pulldown experiment proves that DEPP-N has no interaction with Sedlin,while DEPP,DEPP（ΔPPPSP）and DEPP（S34A）all interact with Sedlin,Immunoprecipitation further proved the interaction between DEPP and Sedlin.Conclusion The domain of Depp and Sedlin interacti on located at the C-terminal of DEPP,and the PPPSP motif on DEPP is independent of the interaction,which lays a foundation for the progressive analysis of the interaction mechanism between D EPP and Sedlin.