Cell proliferation experiment, Immunoprecipitation and western blot Essay

Cell proliferation experiment, Immunoprecipitation and western blot experiment, cell imaging experiment - Essay Example The aim of this work was to elucidate the mechanism by which cp250 causes tumors and identify which signaling pathways were activated by its binding to the cell. The first experiment looked at the effect of cp250 on cell proliferation and compared it to the effect of the growth factor PDGFA and the second and third experiments looked at which pathways were triggered by the binding of cp250 to hepatocytes. The aim of this experiment was to determine whether cp250 caused cell proliferation in hepatocytes and also to establish whether it was recognized by the same receptor that bound the growth factor PDGFA. As shown in Table 1, cp250 and PDGFA clearly have similar effects on cell proliferation. Hepatocytes that were treated with either cp250 or PDGFA both showed an increase in proliferation as compared to cells that were not given either. Moreover cp250 and PDGFA appear to bind to the same receptor, PDGFR. There does not appear to be any synergism when both are used together. Hepatocytes that were treated with antisense RNA to the receptor prior to incubation with the ligands did not show a significant increase in proliferation. In addition, scrambled siRNA that does not target any cellular mRNA does not affect the binding of either cp250 or PDGFA to the receptor. This indicates that the binding of cp250 is specific to PDGFR. The next step was to identify which downstream elements in the signaling process were activated on binding to the receptor. In order to do this, hepatocytes that were treated with cp250 and PDGFA were lysed and complexes that were formed were pulled down with an antibody to the receptor. Western blots with antibodies raised against different downstream signaling molecules were performed to identify which components were complexed with the receptor after activation. Controls were performed with cells treated with water. As can be seen in Fig.1A, cp250 and PDGFA both bind to the receptor PDGFR, confirming the first experiment. From Fig.1B, it can be seen that Phospholipase gamma 1 (PLC1) is recruited to the receptor after treatment with either cp250 or PDGFA. From Fig.1C, it can be inferred that autophosphorylation of PDGFR and phosphorylation of PLC1 take place, indicating the activation of PLC1 takes place once it is recruited to the activated receptor. Confocal Microscopy The aim of this experiment was to confirm the activation of the inositol phospholipids pathway and further characterize the downstream signaling pathway set off by the binding of cp250 to PDGFR. The confocal images show that when rat hepatocytes transfected with EGFP-tagged PLC1 are treated with either cp250 or PDGFA, the PLC1 is localized to the cell membrane, indicating their recruitment. This localization is markedly absent in cells treated with water alone. On addition of the red fluorescent Ca2+ sensing dye X-rhod-1, there is an increase in red fluorescence in the cytoplasm of hepatocytes treated with cp250 or PDGFA but not with water. This indicates that the IP3 signalling pathway has been activated upon binding of the viral protein. DISCUSSION The present work was intended to try and deduce the probable mechanism by which cp250 acts upon the hepatocyte and causes tumorigenesis. Given its similarity to the growth factor PDGFA, it seemed reasonable to postulate that it bound to the same