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. 2015 Mar 19;14(10):1517–1528. doi: 10.1080/15384101.2015.1026519

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© 2015 The Author(s). Published with license by Taylor & Francis Group, LLC

© Sara Badodi, Fiorenza Baruffaldi, Massimo Ganassi, Renata Battini, and Susanna Molinari

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. The moral rights of the named author(s) have been asserted.

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Figure 3. — MEF2C protein level is controlled by CDC20 or CDH1, depending on the phosphorylation of Ser98 and Ser110. (A). MEF2C physically interacts with CDC20 and CDH1. Co-immunoprecipitation analysis of HEK293T cells transiently transfected with empty vector (EV) or HA-tagged vectors coding for CDC20 and CDH1 along with FLAG-MEF2C WT or 2SA. 48 hours after transfection, cells were treated with MG132 and harvested. Total protein extracts (Input) were incubated with protein A beads conjugated with anti-HA antibody. Left: input. Right: immunoprecipitated proteins. FLAG-tagged MEF2C was detected by Western blotting with anti-FLAG antibody. Vinculin was used as a loading control. Asterisk (*) indicates non-specific bands. (B) MEF2C expression is reduced by CDC20 and CDH1 overexpression, CDC20 dependent degradation relies on pSer98/pSer110 phosphorylation. C2 proliferating myoblasts were co-transfected with an empty vector (EV) or HA-tagged CDC20 and CDH1 vectors along with phosphorylable (WT) or not-phosphorylable (2SA) MEF2C. Cell extracts were analyzed by Western blotting with antibodies against FLAG and HA. Vinculin was used as loading control. The histogram (right panel) reports the densitometric quantification of MEF2C protein level (FLAG signal) normalized to the total amount of protein expressed relatively to the quantity of protein in the control sample, taken as 1. Histograms show means ± SEM of 3 independent experiments. ** represent P-values ≤ 0.01. (C) RNAi knockdown of Cdc20 and/or Cdh1 results in an accumulation of MEF2C. Asynchronous C2 cells were transfected with a control siRNA (siCTRL) or a pool of siRNAs targeting Cdh1 (siCdh1, left panel) or Cdc20 (siCdc20, middle panel) mRNAs and analyzed 72 hours later. Alternatively C2 cells transfected with siCdc20 were treated over-night with Nocodazole after 36 hours from transfection (left panel). Protein extracts were analyzed by Western blotting with antibody against MEF2C, CDH1, CDC20 and histone H3 phosphorylated on Ser10, a marker of the M phase. Vinculin was used as loading control. The results of the Western blot shown in A and C are representative of 2 independent experiments that gave similar profiles.