<i>Tb</i>TIF2 is essential for maintaining normal <i>Tb</i>TRF protein levels.

<p>(A) Western blotting was performed with whole cell extracts prepared at various time points after induction of <i>Tb</i>TIF2 RNAi (shown on top). (B) Northern blotting shows that <i>Tb</i>TRF mRNA levels were not affected in <i>Tb</i>TIF2-depleted cells. rRNA precursors were shown as a loading control. (C) Western blotting was performed using whole cell extracts prepared from <i>Tb</i>TIF2 RNAi cells treated with and without MG-132 (Sigma), an inhibitor of the 26S proteasome. <i>Tb</i>AUK1 is normally degraded by the 26S proteasome while <i>Tb</i>PSA6 is not. (D) Western analysis showed that <i>Tb</i>TIF2 protein levels are not sensitive to <i>Tb</i>TRF depletion. In western analyses, <i>Tb</i>TIF2-F2H and <i>Tb</i>AUK1-3HA were detected using an HA monoclonal antibody (F-7, SantaCruz Biotechnology). <i>Tb</i>TRF [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156746#pone.0156746.ref029" target="_blank">29</a>], <i>Tb</i>RAP1 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156746#pone.0156746.ref042" target="_blank">42</a>], <i>Tb</i>PSA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156746#pone.0156746.ref043" target="_blank">43</a>], and tubulin [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156746#pone.0156746.ref044" target="_blank">44</a>] were detected by their respective antibodies. EF-2 was detected using a goat polyclonal antibody against human EF-2 (Santa Cruz Biotechnology). All primary antibodies were diluted 1,000 fold. In this and other figures, proteins from 15 million cells were loaded each lane for western blotting, except when detecting tubulin, proteins from only 0.5 million cells were loaded each lane.