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. 2014 Jan 10;42(6):3565–3579. doi: 10.1093/nar/gkt1371

Figure 4.

Figure 4.

Ectopic NIR stabilizes MDM2 and inhibits MDM2 ubiquitination. (A) NIR causes MDM2 levels to increase independently of p53. H1299 cells were transfected with plasmids producing Flag-NIR (4 µg) and MDM2 (4 µg), and 24 h after transfection, total protein and RNA were extracted. The levels of gapdh and MDM2 transcripts were determined by semi-quantitative RT-PCR (upper panels). Proteins were detected by western blotting and incubation with anti-Flag monoclonal antibody (1:10 000), monoclonal MDM2 antibodies 3G9 (1:2000) or IF2 (1:200), or β-actin antibody (1:10 000). (B) Flag-NIR increases MDM2 levels in U2OS and H1299 cells in a dose-dependent manner. U2OS and H1299 cultures were transfected with plasmids expressing Flag-NIR (0.5, 1.5 or 3.0 µg) and MDM2 (2 µg) as indicated. At 30 h after transfection, total protein was prepared and subjected to standard western blot analysis. Proteins were visualized as in (A), with antibody 3G9 for the detection of MDM2. (C) MDM2 has a longer half-life in the presence of Flag-NIR. H1299 cells were transfected with MDM2 plasmid (0.5 µg) and Flag-NIR plasmid (1.5 µg) as shown. At 24 h after transfection, de novo protein synthesis was blocked by cycloheximide (CHX; 20 µg/ml) for the indicated times (CHX chase) and the levels of MDM2 and Flag-NIR were analysed by western blotting as in (A). (D) Flag-NIR inhibits the ubiquitination of MDM2 in a dose-dependent manner. H1299 cells were transfected for 24 h with the indicated combinations of expression plasmids to produce MDM2 (3 µg), Flag-NIR (0.5, 1.5 or 3.0 µg) and HA-ubiquitin (3 µg), and were then treated with the proteasome inhibitor MG132 (10 µM) for another 4 h. MDM2 was immunoprecipitated with antibody 3G9 from denatured cell extracts. The slower migrating bands indicative of HA-ubiquitinated MDM2 were detected by western blotting with monoclonal anti-HA antibody (1:1000); the remaining signals were detected as in (A). TCL, total cell lysate. (E) Knockdown of endogenous NIR reduces MDM2 protein level. H1299 cells were transfected with control siRNA (c) or a 1:1 mixture of NIR siRNA1 and 2 (40 nM) for 42 h. Total protein extracts were analysed by western blotting with polyclonal anti-NIR antibody 2719 (1:1000), monoclonal MDM2 antibody 3G9 (1:2000) and monoclonal anti-tubulin antibody (1:1000).