Mdm2 associates with proteasomal proteins. (A) 293T cells were transfected with 7.5 μg of a plasmid encoding Myc-tagged Mdm2 and with 7.5 μg of a plasmid encoding the indicated V5-tagged proteins of the 19S complex. IP: Mdm2 was precipitated and associated proteasomal proteins were detected by Western blotting. TCL: Aliquots of cellular lysates were assayed by Western blotting for protein expression. (B) Section I: Mdm2 was precipitated from U2OS cells. Associated S6b protein was detected by Western blotting. Section II: Bacterially expressed and purified GST or S6b fused to GST were mixed with bacterially expressed V5-tagged Mdm2. GST was pulled down and associated Mdm2 was detected by Western blotting. Section III: Mdm2 expressed in insect cells was mixed with partially purified 26S proteasomes. Mdm2 was immunoprecipitated and associated S8 was detected by Western blotting. (C) H1299 cells were transfected with 7 μg of a plasmid encoding Mdm2 and 3 μg of a plasmid encoding V5-tagged S8. Cell extracts were separated by sucrose gradient. Mdm2, V5-S8, endogenous S8, and α7 (20S) were determined by Western blotting. The black lines indicate where two gels were spliced together. (D) Flag-tagged Mdm2 expressed in insect cells was dephosphorylated with λ-phosphatase in the presence and absence of sodium orthovanadate (NaV), phosphorylated with GSK-3 and mixed with 26S proteasomes. Mdm2 was immunoprecipitated and associated proteasomes were detected by Western blotting. (E) H1299 cells were transfected with 1 μg of a plasmid encoding HA-tagged ubiquitin, 7 μg of a plasmid encoding V5 tagged S6b and with 7 μg of a plasmid encoding wild type Myc-Mdm2 or the indicated mutants. Where indicated, MG132 was added 4 h prior to harvest. Mdm2 was precipitated and cell lysates were incubated with UBP1 or mock treated. Associating S6b was detected by Western blotting.

The C-terminus of Mdm2 associates with the proteasome. (A) Bacterially expressed V5-tagged N-terminus (aa 1–200), V5-tagged central domain (aa 200–299) or V5-tagged C-terminal domain (aa 300–491) of Mdm2 were mixed with bacterially expressed and purified GST, S6b fused to GST or S8 fused to GST. GST was pulled down and associated Mdm2 was determined by Western blotting. (B) Schematic drawing of the C-terminal deletion mutants of Mdm2. Bacterially expressed V5-tagged C-terminal domain of Mdm2 and the indicated deletion mutants were mixed with bacterially expressed and purified GST or S6b fused to GST. GST was pulled down and associated C-terminal fragments of Mdm2 were determined by Western blotting. (C) Bacterially expressed C-terminal domain of Mdm2 harboring the indicated mutations were mixed with bacterially expressed and purified GST or S6b fused to GST. GST was pulled down and associated Mdm2 was determined by Western blotting.

An EDY motif is present on Mdm2 and proteasomal proteins. (A) Alignment of sequences containing the EDY motif from proteasomal proteins and Mdm2. (B) Bacterially expressed V5-tagged C-terminus of human Mdm2 (aa 300–491) was mixed with bacterially expressed and purified GST or GST fused to an EDY-containing peptide of Mdm2 (aa 245–264). (C) H1299 cells were transfected with 1 μg of a plasmid encoding His-tagged ubiquitin together with 0.4 μg of a plasmid encoding p53, 1.2 μg of a plasmid encoding Mdm2 and 30 μg of a plasmid encoding thioredoxin or 30 μg of a plasmid encoding an EDY (wt) or EDA (mu) containing peptide of Mdm2 (aa 245–264) fused to thioredoxin (TRX-EDY) or vector DNA for control. Where indicated, cells were treated with 10 μM MG132 for 4 h. Ubiquitylated proteins (ubi-p53) were purified by adsorption to Ni-agarose and p53 was detected by Western blotting. An aliquot of the cells was tested for expression of p53 and Mdm2. Detection of PCNA was used for loading control (TCL: total cell lysate).

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.

The C-terminus of Mdm2 binds to the central domain of Mdm2. (A) Bacterially expressed V5-tagged C-terminus of Mdm2 (aa 300–491) was mixed with bacterially expressed and purified GST or GST-fused to the central domain of Mdm2 (aa 200–299). GST was pulled down with Glutathione sepharose and associated Mdm2 was determined by Western blotting. (B) Bacterially expressed V5-tagged C-terminus of Mdm2 (aa 300–491) was mixed with bacterially expressed and purified GST or GST-fused to the central domain of Mdm2 (aa 200–299) and 26S proteasomes. The proteasomal protein S8 was precipitated and associated C-terminus of Mdm2 was detected by Western blotting.