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. 2012 Mar 1;40(12):5357–5367. doi: 10.1093/nar/gks198

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© The Author(s) 2012. Published by Oxford University Press.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 3. — MDM2 induces ubiquitinylation of TTF-I dependent on its E3 ubiquitin ligase activity. (A) Increasing levels of MDM2 induce TTF-I poly-ubiquitinylation. FLAG-TTF-I, HA-Ubiquitin (HA-Ub) and increasing levels of Myc-MDM2 were co-expressed in HEK293T cells, in the presence of the proteasome inhibitor MG132, and FLAG immunoprecipitates (IP) probed with anti-FLAG and anti-HA antibodies in comparison with the whole-cell lysates. (B) TTF-I ubiquitinylation is abrogated by the C462A point mutation of MDM2 that inactivates the ubiquitin ligase activity (MDM2(C462A)) (22,23). FLAG-TTF-I, HA-Ubiquitin and wild type (WT) or inactive MDM2 (C462A) were co-expressed as in (A) in the presence of MG132, and FLAG immunoprecipitates probed with anti-FLAG and anti-HA antibodies.