Silencing of MAGE-A expression decreases the levels of MDM4 via the proteasome and increase MDM2/MDM4 association.

<p>MAGE-A expression in U2OS cells was silenced by RNAi as previously described or treated with a non-silencing siRNA oligonucleotide as control [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0127713#pone.0127713.ref012" target="_blank">12</a>]. The various proteins were detected by western blotting. (A) Silencing of MAGE-A in U2OS cells. (B) Silencing of MAGE-A was carried out in the absence or presence of the proteasomal inhibitor, bortezomid. The cells were treated with 10 μM bortezomib 6 h prior to harvesting. (C) Co-immunoprecipitation analysis in which MDM2 immunoprecipitates (obtaining using the antibody, 4B2) were probed for MDM4 (8C6) and for MDM2 itself. (D) Cycloheximide-chase analysis of MDM4 in the U2OS cells was carried out following treatment with the non-silencing and MAGE-A-silencing siRNA oligonucleotides. Quantification of the western blots was carried out using ImageJ software. In all cases the data shown are representative of at least three independent experiments.