The stability of endogenous Ci is regulated by the UPS <i>in vivo</i>.

<p>(<b>A</b>–<b>C</b>) UPS inhibition protected CiFL from degradation in the wing disc. CiFL, detected with the 2A1 antibody, accumulated abutting the anterior/posterior (a/p) boundary (marked by the dashed line) of a wildtype wing disc treated with DMSO (<b>A</b>). Incubation with the UPS inhibitor MG132 led to accumulation of CiFL in more anterior cells in the wing disc (<b>B</b>), while the lysosomal inhibitor E64 had no obvious effect (<b>C</b>). (<b>D</b> and <b>E</b>) Blockage of ubiquitination in the wing disc by overexpressing UAS-<i>ubp</i> resulted in accumulation of CiFL in more anterior cells (<b>E</b>). <i>MS1096</i>-Gal4 (G4), which was used in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024168#pone-0024168-g002" target="_blank">Figures 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024168#pone-0024168-g003" target="_blank">3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024168#pone-0024168-g004" target="_blank">4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024168#pone-0024168-g006" target="_blank">6</a> to drive transgene expression at a much higher level in the dorsal compartment of the wing disc (indicated by a box bracket), did not alter the stability of CiFL (<b>D</b>). (<b>F</b>–<b>O</b>) Genetic manipulation to disrupt UPS- or lysosome-mediated protein degradation in wing discs. Knockdown of the 19S proteasome subunit <i>Mov34</i> by RNAi (<b>F</b>) or disrupting the function of the 20S proteasome core particle b2 subunit by expression of a dominant negative temperature-sensitive <i>DTS7</i> transgene (<b>G</b>) in the dorsal compartment of wing discs (indicated by box brackets) resulted in significant accumulation of CiFL. In contrast, the expression pattern of CiFL was not altered when the lysosomal function was disrupted by <i>Hrs</i> RNAi (<b>H</b>, box bracket) or in <i>Hrs<sup>D28</sup></i> loss-of-function somatic clones (<b>J</b>–<b>L</b>, arrowheads). As a control, accumulation of Delta protein (Dl), which normally undergoes endocytosis to the lysosome, was observed when <i>Hrs</i> function was disrupted (<b>I</b>, <b>M</b>–<b>O</b>). <i>Hrs<sup>D28</sup></i> loss-of-function clones were negatively marked by nuclear GFP (nGFP; <b>K</b> and <b>N</b>). Note that the <i>MS1096</i>-Gal4 driver alone did not alter the expression patterns of CiFL or Dl in wing discs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0024168#pone.0024168.s001" target="_blank">Figure S1</a>).</p>