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. Author manuscript; available in PMC: 2010 Aug 14.
Published in final edited form as: Mol Cell. 2009 Aug 14;35(3):316–326. doi: 10.1016/j.molcel.2009.07.014

Figure 2. Ribosomal Protein S7 Stabilizes p53 and Inhibits MDM2-mediated p53 Polyubiquitination.

Figure 2

(A) U2OS cells were transfected with HA-p53 (0.35 µg), Flag-MDM2 (1.5 µg), and Myc-S7 (1.5 µg) constructs as indicated. Cell lysates were used for IB with indicated antibodies. GFP construct was added for transfection and loading control. (B) Ectopic expression of S7 stabilizes endogenous MDM2 and p53. U2OS cells were transfected with increasing amounts of Myc-S7 (0.5, 1, 2, and 3 µg) as indicated. Cell lysates were subjected to IB with indicated antibodies. (C) Ectopic expression of S7 stabilizes exogenous MDM2 independently of p53. H1299 cells were transfected with increasing amount of Myc-S7 (0.5, 1, 2 µg) or pcDNA3-MDM2 (1, 2, 3 µg) as indicated. Cell lysates were used for IB with indicated antibodies. (D) Ectopic expression of S7 inhibits MDM2-mediated p53 polyubiquitination and MDM2 autoubiquitination. H1299 cells were transfected with combinations of Myc-S7 (1.2 µg), Myc-S7Δ (1.2 µg), and Flag-MDM2 (1.2 µg) plasmids with (left panel) or without (right panel) p53 (0.3 µg) plasmid in the presence of an HA-Ub (1.2 µg) plasmid as indicated. The cells were treated with MG132 (20 µM) for 4 hours before harvesting. Whole-cell lysates were subjected to IP with anti-p53 (FL393-G) antibody followed by IB with anti-HA antibody to detect ubiquitinated p53 (left panel) or IP with anti-Flag antibody followed by IB with anti-HA antibody to detect ubiquitinated MDM2 (right panel).