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. 2000 Feb;20(4):1243–1253. doi: 10.1128/mcb.20.4.1243-1253.2000

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Copyright © 2000, American Society for Microbiology

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FIG. 4 — Identification of the region from amino acids 92 to 112 of p53 as an essential element for Mdm2-mediated degradation. (A) The p53-p73 chimeras with a more refined swapping at the PRD were prepared as described in Fig. 1 by switching each segment between p53 and p73 at the indicated position. (B) p21 levels are induced by the expression of the chimeras. A vector (2.5 μg) containing the indicated cDNA was transfected into Saos-2 cells, and the transfectants were analyzed by immunoblotting (IB) with anti-p21 (upper panel) and anti-Flag (lower panel) 24 h posttransfection. (C) The segment from amino acids 92 to 112 of p53 is essential for Mdm2-mediated degradation. The p53-p73 chimeras with more refined swapping at the PRD were tested for sensitivity to Mdm2-mediated degradation as described for Fig. 2. Levels of the chimeras and transfection efficiency were determined by Western analysis with anti-Flag (upper panel) and anti-GFP (lower panel) respectively. (D) The p53-p73 chimeras bind to Mdm2 with an affinity comparable to that of their wild-type counterparts. Flag-tagged vectors expressing wild-type p53, wild-type p73, or the indicated chimeras were coexpressed with pCMV-Mdm2. Cell lysates were prepared 24 h posttransfection and subjected to anti-Flag immunoprecipitation. The immunocomplexes were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and analyzed by immunoblotting with anti-Mdm2 (upper panel) and anti-Flag (lower panel). Flag-tagged Rad52 was included as a control. (E) The p53 mutant lacking amino acids 92 to 112 is no longer sensitive to Mdm2-mediated degradation. The p53(Δaa92-112) mutant prepared by a two-step PCR was tested for sensitivity to Mdm2-mediated degradation as described in the legend to Fig. 2. The corresponding deletion mutant of p73 was included as a control. Levels of the proteins and transfection efficiency were determined by Western analysis with anti-Flag (upper panel) and anti-GFP (lower panel), respectively. (F) The p53 or p73 deletion mutants retain the ability to bind to Mdm2. Association of the deletion mutants with Mdm2 was examined as described for panel D.

FIG. 4 — Identification of the region from amino acids 92 to 112 of p53 as an essential element for Mdm2-mediated degradation. (A) The p53-p73 chimeras with a more refined swapping at the PRD were prepared as described in Fig. 1 by switching each segment between p53 and p73 at the indicated position. (B) p21 levels are induced by the expression of the chimeras. A vector (2.5 μg) containing the indicated cDNA was transfected into Saos-2 cells, and the transfectants were analyzed by immunoblotting (IB) with anti-p21 (upper panel) and anti-Flag (lower panel) 24 h posttransfection. (C) The segment from amino acids 92 to 112 of p53 is essential for Mdm2-mediated degradation. The p53-p73 chimeras with more refined swapping at the PRD were tested for sensitivity to Mdm2-mediated degradation as described for Fig. 2. Levels of the chimeras and transfection efficiency were determined by Western analysis with anti-Flag (upper panel) and anti-GFP (lower panel) respectively. (D) The p53-p73 chimeras bind to Mdm2 with an affinity comparable to that of their wild-type counterparts. Flag-tagged vectors expressing wild-type p53, wild-type p73, or the indicated chimeras were coexpressed with pCMV-Mdm2. Cell lysates were prepared 24 h posttransfection and subjected to anti-Flag immunoprecipitation. The immunocomplexes were resolved by SDS-PAGE, transferred onto a nitrocellulose membrane, and analyzed by immunoblotting with anti-Mdm2 (upper panel) and anti-Flag (lower panel). Flag-tagged Rad52 was included as a control. (E) The p53 mutant lacking amino acids 92 to 112 is no longer sensitive to Mdm2-mediated degradation. The p53(Δaa92-112) mutant prepared by a two-step PCR was tested for sensitivity to Mdm2-mediated degradation as described in the legend to Fig. 2. The corresponding deletion mutant of p73 was included as a control. Levels of the proteins and transfection efficiency were determined by Western analysis with anti-Flag (upper panel) and anti-GFP (lower panel), respectively. (F) The p53 or p73 deletion mutants retain the ability to bind to Mdm2. Association of the deletion mutants with Mdm2 was examined as described for panel D.