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. 2013 May 13;288(26):18939–18946. doi: 10.1074/jbc.M113.454470

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 3. — The extent of polyubiquitination, not monoubiquitination, regulates Mdm2 E3 activity. A and B, ubiquitination of p53 with GST-Mdm2 (A) and HA-Mdm2 (B) that has been auto-ubiquitinated for the indicated times. Ubiquitination of p53 was detected with anti-p53 antibody (top), and Mdm2 auto-ubiquitination was analyzed with anti-Mdm2 (bottom) and anti-ubiquitin (middle) antibodies. C, ubiquitination of p53 in the presence of no Mdm2 (−), unmodified Mdm2 (−Ub), mono-ubiquitinated Mdm2 (Me Ub), or polyubiquitinated Mdm2 (WT Ub). Mdm2 auto-ubiquitination was probed with anti-Mdm2 (bottom left) and anti-ubiquitin (top left). Ubiquitinated p53 was detected with anti-p53 antibody (right).