USP25 and HRD1 have opposing effects on CD3

Abstract

<p>δ <b>protein levels and ubiquitination.</b> A) HEK-293 cells were transfected as indicated and harvested 48 hours later. Western blots are from whole cell lysates. HRD1(WT): normal HRD1; HRD1(CA): catalytically inactive HRD1, in which the catalytic cysteine is substituted by an alanine residue <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#pone.0036542-Kikkert1" target="_blank">[7]</a>. Histograms on the right: semi-quantification of data from the left and other independent experiments. Shown are means +/− standard deviations. CD3δ levels were normalized to loading control. P values from Student T tests are shown below histograms. B and C) HEK-293 cells were transfected with the indicated constructs. 48 hours post transfection, cells were treated for 6 hours with MG132 (15 µM) and HA-CD3δ was immunopurified using bead-bound anti-HA antibody after a stringent denature/renature step (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036542#s4" target="_blank">Materials and Methods</a> for details). Histograms: semi-quantification of bracketed ubiquitin smears from the experiment on the left and other similar, independent experiments. Shown are means +/− standard deviations. P values for panel C are from Student T-tests. </p