Fig. 6

Upregulation of Mad1 prevents the interaction between MDM2 and PML, freeing MDM2 to bind p53. a Increased expression of full length (FL) Mad1 but not Mad1 lacking the CTD (aa 1–596; Mad1ΔCTD) impairs co-immunoprecipitation of PML with MDM2. 293T cells were co-transfected with HA-PML, 3xFLAG-MDM2, and increasing amounts of FL Mad1 or Mad1ΔCTD. b Reciprocal experiment showing that increased expression of FL Mad1 but not Mad1ΔCTD impairs co-precipitation of MDM2 with PML. 293T cells were co-transfected with 3xFLAG-PML, 2xHA-MDM2, and increasing amounts of FL Mad1 or Mad1ΔCTD. c Upregulation of Mad1 inhibits the interaction of MDM2 with PML, freeing MDM2 to interact with p53. 293T cells were co-transfected with 2xHA-PML, 3xFLAG-MDM2, and increasing amounts of FL Mad1 or Mad1ΔCTD. a–c 48 h after transfection, cell extracts were prepared and immunoprecipitated using beads coupled to anti-FLAG antibodies. Blots are representative of 3 independent experiments