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. 2006 Oct 23;27(1):111–119. doi: 10.1128/MCB.00235-06

FIG. 4.

FIG. 4.

Modulation of abundance of the p90MDM2 and p75MDM2 isoforms and of p53 and TSG101 by the p53/MDM2 and MDM2/TSG101 control loops. (A) Effect of p90MDM2 and p75MDM2 on p53 protein abundance. Saos-2 cells were cotransfected with a construct expressing p53 (25 ng) and with vectors expressing HA-tagged MDM2 proteins, including full-length (HA-MDM2-FL) and N-terminal truncation proteins as indicated by the position of the last amino acid in the deleted portion. HA-tagged MDM2, p53, and β-tubulin were detected by immunoblotting (IB) using anti-HA, anti-p53, and anti-β-tubulin antibodies as indicated. (B) Physical interaction of p90MDM2 and p75MDM2 with TSG101. The indicated constructs were introduced into Saos-2 cells, and protein extracts from these cells were subjected to immunoprecipitation with anti-TSG101 antibody, as indicated by the designation IP. Coprecipitated proteins were examined by immunoblotting with antibodies against TSG101 or HA epitope. (C) Effect of TSG101 expression on the 26S proteasome-mediated degradation of MDM2. U2OS cells were cotransfected with MDM2 constructs expressing full-length as well as N-terminally truncated proteins. Forty-eight hours posttransfection, cells were left untreated or were treated with proteasome inhibitor MG132 (40 uM) for another 8 h, and cellular protein extracts were analyzed by immunoblotting (lanes 1, 2, 4, and 6). The indicated MDM2 constructs and a construct expressing HA-tagged TSG101 were cointroduced into U2OS cells by transfection (lanes 5 and 7). HA-tagged MDM2 as well as TSG101, β-tubulin, and GFP were detected by immunoblotting with anti-HA, anti-β-tubulin, and anti-GFP antibodies, respectively. An empty vector was introduced into cells as a control (lane 3). To ensure comparable transfection efficiency among these samples, a GFP expression construct, pCMV-GFP (0.1 μg), was included, and its protein level is shown in the bottom panel. (D) Effect of p90MDM2 and p75MDM2 on the protein stability of TSG101. A constant amount of HA-tagged TSG101 construct (0.5 μg) was cotransfected with constructs expressing HA-tagged MDM2 isoforms in Saos-2 cells. Cellular protein levels of HA-MDM2 and HA-TSG101 were detected by immunoblotting with anti-HA antibody. (E) Model showing the proposed role of p90MDM2 in the p53/MDM2 and MDM2/TSG101 feedback control loops.