Beads were collected by centrifugation, and the supernatants were transferred to new tubes containing agaroseconjugated antibodies to either p or mdm. Lysates were rotated overnight at C, and proteinantibodybead complexes were washed three times in l of lysis buffer.Plasmids containing the CAT gene driven by two copies of the consensus presponsive element in ml of serumfree medium, and the mixture was allowed to stand for min.The mixture was then added to cells and left for hbefore addition of ml of normal complete medium.Cells were incubated overnight at C in a humidified incubator before exposure to M bortezomib or M etoposide for h.Thujone samples were then mixed with l of a chloramphenicol preparation and incubated at C for h.One ml of ethyl acetate was added to the reaction, and samples were vortexed vigorously and centrifuged.A total volume of l of the upper layer was transferred to another tube and dried under vacuum was added to each tube before l were added dropwise to a silica gel sheet. The sheet was placed in a chamber and resolved in methanol and chloroform until the solvent front came to within cm of the top.The membrane was then air dried and placed against a phosphorimaging cassette.Complexes were stabilized by incubation with l of antip antibody at C for min.Complexes were then loaded onto acrylamide gels, resolved by electrophoresis, and dried.ProteinDNA complexes were detected by autoradiography.Cells were split to well plates and left to recover for h.Cells were then treated with M bortezomib or M etoposide for hbefore harvest by trypsinization.Growth and wash media were saved along with trypsinized cells, and samples were centrifuged at gfor min.Supernatants were removed, and pellets were resuspended in l of a propidium iodide solution. Samples were stored at C for hbefore analysis by flow cytometry.The cells with subdiploid DNA content were quantified to determine the percentages of cells containing fragmented DNA.The representative blots shown are typical of three independent experiments.Proteasome inhibitorstabilized p was predominately nuclear and displayed a distribution pattern comparable with that observed with etoposide. Recent work also suggests that p can translocate to mitochondria in cells undergoing apoptosis. The lack of phosphorylation on sites associated with interactions with mdm prompted us to investigate the interaction between p and mdm in cells exposed to bortezomib.In both cases, we found that mdm remained bound to p after bortezomib treatment, whereas pmdm complexes were much less prominent in cells exposed to etoposide. These lysates were then subjected to immunoblot analysis for p, p on serine, or actin.Representative blots shown are typical of three independent experiments.Phosphorylation of p on serines and is associated with the release of p from mdm and transcriptional activation.Representative images displayed are typical of results obtained in three independent experiments.These were then immunoprecipitated conjugated to agarose beads.The immunoprecipitated complexes resolved by SDSPAGE and p and mdm were detected by immunoblotting.Whole cell lysates from the same samples were used to monitor total cellular Flunisolide levels of each protein.Representative blots typical of three independent experiments are shown.An antibody specific to p was used to stabilize any proteinDNA complexes that were formed as reported previously.