Dayle Parnell-Lampen (Providence) and Kirsten Prufer, Biological Sciences

Developing an immunoprecipitation method to identify nuclear transport protein interactions of LXRα and LXRβ


LXRα and LXRβ are nuclear receptor proteins involved in cholesterol metabolism. The precise mechanism of the nuclear transport of these proteins is not yet understood. In this experiment, the objective was to better understand this transport by developing a method to identify which nuclear transport proteins bind to LXRα and LXRβ in the cytoplasm and in the nucleus. 293 cells used in this experiment contained plasmids bearing a gene for YFP spliced next to a gene for either LXRα or LXRβ so that the genes would be translated into a single YFP-LXR protein. Once enough protein was synthesized, two different extraction methods were used. A cellular fractionation using a Dounce homogenizer was done to isolate nuclear and cytoplasmic extracts and RIPA whole cell extracts were also made. These extracts were immunoprecipitated with a GFP antibody and a rabbit IgG antibody as a control. Western blot analysis was performed on the isolated proteins and staining was done for RXR. RXR was chosen because it is a heterodimer of LXR and is expected to interact very strongly with LXR. The gels consistently showed bands at the molecular weight of RXR but the protein did not appear to be specifically pulled down by the GFP antibody. Extracts that had not been subjected to immunoprecipitation were also stained for RXR as an additional control. While the fractionation method using the Dounce homogenizer showed RXR staining, the RIPA extracts did not. This indicates that the RIPA extraction method is unsuitable for this procedure as it probably causes degradation of the proteins. Overall, these results indicate that this method was unsuccessful. An optimized immunoprecipitation method, using the cellular fractionation by Dounce homogenizer technique, will need to be developed in order to identify the nuclear transport protein interactions of LXRα and LXRβ