Figure 7. TBP-1 is a downstream target of MDM2 activation.

A: U2OS cells were either transfected (lanes +) or untransfected (lanes −) with the MDM2 expression plasmid. 24 hrs after transfection cell extract was prepared and subjected either to immunoprecipitation with anti-TBP-1 antibody where indicated or, with anti-GFP antibody as negative control. Cell extracts were also incubated with protein A-agarose as control, where indicated. Immunoprecipitated extracts were analyzed by Western Blot with anti-MDM2 or anti-TBP-1 antibody. Aliquots of cell extracts were analyzed by Western Blot before immunoprecipitation (input). B: U2OS cells were either transfected (lanes +) or untransfected (lanes −) with the MDM2 expression plasmid. 24 hrs after transfection cell extract was prepared and subjected either to immunoprecipitation with anti-MDM2 antibody where indicated or, with anti-Flag antibody as negative control. Cell extracts were also incubated with protein A-agarose as control, where indicated. Immunoprecipitated extracts were analyzed by Western Blot with anti-MDM2 or anti-TBP-1 antibody. Aliquots of cell extracts were analyzed by Western Blot before immunoprecpitation (input). C: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of MDM2 expression plasmid. After 24 hrs, cells were lysed and whole cell extracts probed with anti-TBP-1, anti-MDM2, and anti-actin, for loading control. D: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of MDM2 expression plasmid. After 24 hrs cells were treated either with DMSO (first four lanes) or with 10 µM MG132 where indicated. Cell extracts were analyzed by Western Blot with anti-Xpress (to reveal transfected TBP-1), anti-MDM2, and anti-actin, for control. E: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of either MDM2_wt, MDM2_S166A or MDM2_S166A/S186A expression plasmids. After 24 hrs cells were lysed and whole cell extracts were analyzed by Western Blot with anti-Xpress (to reveal transfected TBP-1), anti-MDM2, and anti-actin, for control. F: U2OS cells were transfected with TBP-1 expression plasmid and increasing amounts of either MDM2_wt, MDM2_1–441 or MDM2_Δ150–230 expression plasmids. After 24 hrs cells were lysed and whole cell lysates analyzed by Western Blot with anti-Xpress (to reveal transfected TBP-1), anti-MDM2, and anti-actin, for control.