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. 2010 May 17;107(22):10038–10043. doi: 10.1073/pnas.0911716107

Fig. 5.

Fig. 5.

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).