Fig. 2.

p53, Mdm2 and the proteasome form a ternary complex. (A) H1299 cells were transfected with 5 μg of a plasmid encoding p53 together with 5 μg of a plasmid encoding Mdm2 and with a plasmid encoding Flag-tagged S6b, where the amount of transfected plasmid was adjusted to receive equal levels. 4 h prior to harvest, 10 μM MG132 were added. IP:α-Flag: Flag-tagged S6b was precipitated and associated p53 and Mdm2 were determined by Western blotting. TCL: 50 μg of total cell lysate were separated on a SDS-PAGE gel. Mdm2, p53 and S5a were determined by Western blotting. (B) H1299 cells were transfected with 5 μg of a plasmid encoding p53 or with vector together with 5 μg of a plasmid encoding wild type Mdm2, the indicated mutants of Mdm2 or vector DNA and with 5 μg of a plasmid encoding V5-tagged S5a. 4 h prior to harvest, MG132 was added. (B) Section I: IP:α-p53: p53 was precipitated and associated S5a and Mdm2 were determined by Western blotting. TCL: 50 μg of total cell lysate were separated on a SDS-PAGE gel. Mdm2, p53 and S5a were determined by Western blotting. Section II: The signals for p53 and S5a from IP and TCL were quantified and the ratios between p53 and S5a were calculated. Mean values and standard deviations of the relative amount of S5a bound to p53 of three independent experiments were plotted. p53 bound to S5a in the presence of wt Mdm2 was set to 1. (C) U2OS cells were transfected with siRNA targeted against Mdm2 or with a control siRNA. 10 μM MG132 were added 4 h prior to harvest. P53 was precipitated and associated S8 was determined by Western blotting.