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. Author manuscript; available in PMC: 2015 Jul 22.
Published in final edited form as: Mol Cell. 2010 Sep 24;39(6):963–974. doi: 10.1016/j.molcel.2010.08.029

Figure 5. MAGEs Enhance the Ubiquitin Ligase Activity of E3 RING Proteins.

Figure 5

(A) MAGE-G1 enhances NSE1 ubiquitin ligase activity in vitro. Biotin-ubiquitin, ubiquitin E1, UbcH13/MMS2 E2, Mg-ATP, and the indicated MAGE-RING proteins were incubated for 60 min at 37°C. Biotin-ubiquitin was detected by Streptavidin-HRP.

(B) Mutation of the dileucine motif within MAGE-G1 WH-A disrupts binding to the NSE1 RING protein, but not NSE4a. The indicated GST-tagged MAGE-G1 proteins were incubated with in vitro translated Myc-NSE1 or Myc-NSE4a. Proteins were detected by anti-Myc and anti-GST immunoblotting.

(C) MAGE-C2 enhances TRIM28 ubiquitin ligase activity in vitro. FLAG-ubiquitin, ubiquitin E1, UbcH2 E2, Mg-ATP, and the indicated MAGE-RING proteins were incubated for 60 min at 37°C. FLAG-ubiquitin was detected by anti-FLAG immunoblotting.

(D) MAGE-C2 enhances TRIM28 autoubiquitylation in vitro. Reactions were performed as in (C) except TRIM28 was detected by anti-TRIM28 immunoblotting.

(E) MAGE-A2 and -C2 enhance p53 ubiquitylation by TRIM28 in vitro. Reactions were performed as in (A) except substrate (GST-p53 or negative controls GST-MMS21 AA1-165 or free GST) was added and detected by anti-GST immunoblotting. MAGE-G1-NSE1 complex was included as an irrelevant MAGE-RING ligase that does not target p53.

(F) MAGE-C2-induced p53 ubiquitylation requires a functional TRIM28 RING domain and the ability to bind TRIM28. Reactions were performed as described in (E).